Multiplexed analysis of clinical specimens apparatus and methods

ABSTRACT

A method for the multiplexed diagnostic and genetic analysis of enzymes, DNA fragments, antibodies, and other biomolecules comprises the steps of constructing an appropriately labeled beadset, exposing the beadset to a clinical sample, and analyzing the combined sample/beadset by flow cytometry is disclosed. Flow cytometric measurements are used to classify, in real-time, beads within an exposed beadset and textual explanations, based on the accumulated data obtained during real-time analysis, are generated for the user. The inventive technology enables the simultaneous, and automated, detection and interpretation of multiple biomolecules or DNA sequences in real-time while also reducing the cost of performing diagnostic and genetic assays.

[0001] Microfiche appendix A contains a listing of selected Visual Basicand C programming source code in accordance with the inventivemultiplexed assay method. Microfiche appendix A, comprising 1 sheethaving a total of 58 frames, contains material which is subject tocopyright protection. The copyright owner has no objection to thefacsimile reproduction by anyone of the patent disclosure, as it appearsin the Patent and Trademark Office patent files or records, butotherwise reserves all copyright rights whatsoever.

[0002] The invention relates generally to laboratory diagnostic andgenetic analysis and, more particularly, to a flow cytometric method forthe simultaneous and multiplexed diagnostic and genetic analysis ofclinical specimens.

[0003] Analysis of clinical specimens is important in science andmedicine. A wide variety of assays to determine qualitative and/orquantitative characteristics of a specimen are known in the art.Detection of multiple analytes, or separately identifiablecharacteristics of one or more analytes, through single-step assayprocesses are presently not possible or, to the extent possible, haveprovided only very limited capability and have not yielded satisfactoryresults. Some of the reasons for these disappointing results include theextended times typically required to enable the detection andclassification of multiple analytes, the inherent limitations of knownreagents, the low sensitivities achievable in prior art assays whichoften lead to significant analytical errors and the unwieldy collection,classification, and analysis of prior art algorithms vis a vis the largeamounts of data obtained and the subsequent computational requirementsto analyze that data.

[0004] Clearly, it would be an improvement in the art to have adequateapparatus and methods for reliably performing real-time multipledeterminations, substantially simultaneously, through a single orlimited step assay process. A capability to perform simultaneous,multiple determinations in a single assay process is known as“multiplexing” and a process to implement such a capability is a“multiplexed assay.”

[0005] Flow Cytometry

[0006] One well known prior art technique used in assay procedures forwhich a multiplexed assay capability would be particularly advantageousis flow cytometry. Flow cytometry is an optical technique that analyzesparticular particles in a fluid mixture based on the particles' opticalcharacteristics using an instrument known as a flow cytometer.Background information on flow cytometry may be found in Shapiro,“Practical Flow Cytometry,” Third Ed. (Alan R. Liss, Inc. 1995); andMelamed et al., “Flow Cytometry and Sorting,” Second Ed. (Wiley-Liss1990), which are incorporated herein by reference.

[0007] Flow cytometers hydrodynamically focus a fluid suspension ofparticles into a thin stream so that the particles flow down the streamin substantially single file and pass through an examination zone. Afocused light beam, such as a laser beam illuminates the particles asthey flow through the examination zone. Optical detectors within theflow cytometer measure certain characteristics of the light as itinteracts with the particles. Commonly used flow cytometers such as theBecton-Dickinson Immunocytometry Systems “FACSCAN” (San Jose, Calif.)can measure forward light scatter (generally correlated with therefractive index and size of the particle being illuminated), side lightscatter (generally correlated with the particle's size), and particlefluorescence at one or more wavelengths. (Fluorescence is typicallyimparted by incorporating, or attaching a fluorochrome within theparticle.) Flow cytometers and various techniques for their use aredescribed in, generally, in “Practical Flow Cytometry” by Howard M.Shapiro (Alan R. Liss, Inc., 1985) and “Flow Cytometry and Sorting,Second Edition” edited by Melamed et al. (Wiley-Liss, 1990).

[0008] One skilled in the art will recognize that one type of “particle”analyzed by a flow cytometer may be man-made microspheres or beads.Microspheres or beads for use in flow cytometry are generally known inthe art and may be obtained from manufacturers such as Spherotech(Libertyville, Ill.), and Molecular Probes (Eugene, Oreg.).

[0009] Although a multiplexed analysis capability theoretically wouldprovide enormous benefits in the art of flow cytometry, very littlemultiplexing capability has been previously achieved. Prior multiplexedassays have obtained only a limited number of determinations. A reviewof some of these prior art techniques is provided by McHugh, “FlowMicrosphere Immunoassay for the Quantitative and Simultaneous Detectionof Multiple Soluble Analytes,” in Methods in Cell Biology, 42, Part B,(Academic Press, 1994). For example, McHugh et al., “Microsphere-BasedFluorescence Immunoassays Using Flow Cytometry Instrumentation,” inClinical Flow Cytometry Ed. K. D. Bauer, et al., Williams and Williams,Baltimore, Md., 1993, 535-544, describe an assay where microspheres ofdifferent sizes are used as supports and the identification ofmicrospheres associated with different analytes was based ondistinguishing a microsphere's size. Other references in this areainclude Lindmo, et al., “Immunometric Assay by Flow Cytometry UsingMixtures of Two Particle Types of Different Affinity,” J. Immun. Meth.,126, 183-189 (1990); McHugh, “Flow Cytometry and the Application ofMicrosphere-Based Fluorescence Immunoassays,” Immunochemica, 5, 116(1991); Horan et al., “Fluid Phase Particle Fluorescence Analysis:Rheumatoid Factor Specificity Evaluated by Laser Flow Cytophotometry” inImmunoassays in the Clinical Laboratory, 185-198 (Liss 1979); Wilson etal., “A New Microsphere-Based Immunofluorescence Assay Using FlowCytometry,” J. Immunological Methods, 107, 225-230 (1988); and Fulwyleret al., “Flow Microsphere Immunoassay for the Quantitative andSimultaneous Detection of Multiple Soluble Analytes,” Meth. Cell Biol.,33, 613-629 (1990).

[0010] The above cited methods have been unsatisfactory as applied toprovide a fully multiplexed assay capable of real-time analysis of morethan a few different analytes. For example, certain of the assay methodsreplaced a single ELISA procedure with a flow cytometer-based assay.These methods were based on only a few characteristics of the particlesunder analysis and enabled simultaneous determination of only a very fewanalytes in the assay. Also, the analytic determinations made werehampered due to software limitations including the inability to performreal-time processing of the acquired assay data. In summary, although ithas been previously hypothesized that flow cytometry may possibly beadapted to operate and provide benefit in a multiple analyte assayprocess, such an adaptation has not in reality been accomplished.

[0011] Analysis of Genetic Information

[0012] The availability of genetic information and association ofdisease with mutation(s) of critical genes has generated a rich field ofclinical analysis. In particular, the use polymerase chain reaction(PCR) and its variants have facilitated genetic analysis. A majoradvance in this field is described in our co-pending andcontemporaneously filed U.S. Application entitled “Methods andCompositions for Flow Cytometric Determination of DNA Sequences.” Thisco-pending application describes a powerful flow cytometric assay forPCR products, which may be multiplexed in accordance with the presentinvention. A multiplexed flow cytometric assay for PCR reaction productswould provide a significant advantage in the field of genetic analysis.

[0013] Recent advances in genetic analyses have provided a wealth ofinformation regarding specific mutations occurring in particular genesin given disease states. Consequently, use of an individual's geneticinformation in diagnosis of disease is becoming increasingly prevalent.Genes responsible for disease have been cloned and characterized in anumber of cases, and it has been shown that responsible genetic defectsmay be a gross gene alteration, a small gene alteration, or even in somecases, a point mutation. There are a number of reported examples ofdiseases caused by genetic mutations. Testing of gene expression byanalysis of cDNA or mRNA, and testing of normal genes and alleles, as incases of tissue typing and forensics, are becoming widespread. Otheruses of DNA analysis, for example in paternity testing, etc., are alsoimportant and can be used in accordance with the invention.

[0014] Current techniques for genetic analysis have been greatlyfacilitated by the development and use of polymerase chain reaction(PCR) to amplify selected segments of DNA. The power and sensitivity ofthe PCR has prompted its application to a wide variety of analyticalproblems in which detection of DNA or RNA sequences is required.

[0015] PCR is capable of amplifying short fragments of DNA, providingshort (20 bases or more) nucleotides are supplied as primers. Theprimers anneal to either end of a span of denatured DNA target and, uponrenaturation, enzymes synthesize the intervening complementary sequencesby extending the primer along the target strand. During denaturation,the temperature is raised to break apart the target and newlysynthesized complementary sequence. Upon cooling, renaturation andannealing, primers bind to the target and the newly made opposite strandand now the primer is extended again creating the complement. The resultis that in each cycle of heating and renaturation followed by primerextension, the amount of target sequence is doubled.

[0016] One major difficulty with adoption of PCR is the cumbersomenature of the methods of analyzing the reaction's amplified DNAproducts. Methods for detecting genetic abnormalities and PCR productshave been described but they are cumbersome and time consuming. Forexample, U.S. Pat. No. 5,429,923 issued Jul. 4, 1995 to Seidman, et al.,describes a method for detecting mutations in persons having, orsuspected of having, hypertrophic cardiomyopathy. That method involvesamplifying a DNA sequence suspected of containing the disease associatedmutation, combining the amplified product with an RNA probe to producean RNA-DNA hybrid and detecting the mutation by digesting unhybridizedportions of the RNA strand by treating the hybridized product with anRNAse to detect mutations, and then measuring the size of the productsof the RNAse reaction to determine whether cleavage of the RNA moleculehas occurred.

[0017] Other methods used for detecting mutations in DNA sequences,including direct sequencing methods (Maxim and Gilbert, Proc. Natl.Acad. Sci. U.S.A., 74, 560-564, 1977); PCR amplification of specificalleles, PASA (Botttema and Sommer, Muta. Res., 288, 93-102, 1993); andreverse dot blot method (Kawasaki, et al., Methods in Enzymology, 218,369-81, 1993) have been described. These techniques, while useful, aretime consuming and cumbersome and for that reason are not readilyadaptable to diagnostic assays for use on a large scale.

[0018] At least one use of flow cytometry for the assay of a PCR producthas been reported but that assay has not been adapted to multiplexing.See Vlieger et al., “Quantitation of Polymerase Chain Reaction Productsby Hybridization-Based Assays with Fluorescent Colorimetric, orChemiluminescent Detection,” Anal. Biochem., 205, 1-7 (1992). In Vliegeret al. a PCR product was labeled using primers that containedbiotinylated nucleotides. Unreacted primers were first removed and theamplified portion annealed with a labeled complementary probe insolution. Beaded microspheres of avidin were then attached to theannealed complementary material. The avidin beads bearing the annealedcomplementary material were then processed by a flow cytometer. Theprocedure was limited, inter alia, in that avidin beads having only asingle specificity were employed. Further, real-time analysis of theassay's data was not possible.

[0019] Data Manipulation

[0020] The large volume of data typically generated during flowcytometric multiple analyte assays, combined with the limitedcapabilities of prior techniques to collect, sort and analyze such datahave provided significant obstacles in achieving a satisfactorymultiplexed assay. The computing methods used in prior art flowcytometric analyses have generally been insufficient and unsuited foraccurately and timely analyzing large volumes of data such as would begenerated by multiplexed assays; particularly when more than twoanalytes (or properties of a single analyte) are to be simultaneouslydetermined.

[0021] The present invention enables the simultaneous determination ofmultiple distinct analytes to a far greater degree than existingtechniques. Further, the invention provides an improved dataclassification and analysis methodology that enables the meaningfulanalysis of highly multiplexed assays in real-time. The invention isbroadly applicable to multiplexed analysis of a number of analytes in ahost of bioassays in which there is currently a need in the art.

[0022] The present invention provides improved methods, instrumentation,and products for detecting multiple analytes in a fluid sample by flowcytometric analysis and for analyzing and presenting the data inreal-time. An advantage of the invention is that it allows one rapidlyand simultaneously to detect a wide variety of analytes of interest in asingle assay step.

[0023] The invention employs a pool of bead subsets. The individualsubsets are prepared so that beads within a subset are relativelyhomogeneous but differ in at least one distinguishing characteristicfrom beads in any other subset. Therefore, the subset to which a beadbelongs can readily be determined after beads from different subsets arepooled.

[0024] In a preferred embodiment, the beads within each subset areuniform with respect to at least three and preferably four knownclassification parameter values measured with a flow cytometer: e.g.,forward light scatter (C₁) which generally correlates with size andrefractive index; side light scatter (C₂) which generally correlateswith size; and fluorescent emission in at least one wavelength (C₃), andpreferably in two wavelengths (C₃ and C₄), which generally results fromthe presence of fluorochrome(s) in or on the beads. Because beads fromdifferent subsets differ in at least one of the above listedclassification parameters, and the classification parameters for eachsubset are known, a bead's subset identity can be verified during flowcytometric analysis of the pool in a single assay step and in real-time.

[0025] Prior to pooling subsets of beads to form a beadset, the beadswithin each subset can be coupled to a reactant that will specificallyreact with a given analyte of interest in a fluid sample to be tested.Usually, different subsets will be coupled to different reactants so asto detect different analytes. For example, subset 1 may be labeled so asto detect analyte A (AnA); subset 2 may be labeled so as to detectanalyte B (AnB); etc.

[0026] At some point prior to assay, the variously labeled subsets arepooled. The pooled beads, or beadset, are then mixed with a fluid sampleto test for analytes reactive with the various reactants bound to thebeads. The system is designed so that reactions between the reactants onthe bead surfaces and the corresponding analytes in the fluid samplewill cause changes in the intensity of at least one additionalfluorescent signal (F_(m)) emitted from a fluorochrome that fluorescesat a wavelength distinct from the wavelengths of classificationparameters C₃ or C₄. The F_(m) signal serves as a “measurement signal,”that is, it indicates the extent to which the reactant on a given beadhas undergone a reaction with its corresponding analyte. The F_(m)signal may result from the addition to the assay mixture offluorescently labeled “secondary” reagent that binds to the bead surfaceat the site where a reactant-analyte reaction has occurred.

[0027] When the mixture (pooled beads and fluid sample) is run through aflow cytometer, each bead is individually examined. The classificationparameters, e.g., C₁, C₂, C₃, and C₄, are measured and used to classifyeach bead into the subset to which it belongs and, therefore, identifythe analyte that the bead is designed to detect. The F_(m) value of thebead is determined to indicate the concentration of analyte of interestin the fluid sample. Not only are many beads from each subset rapidlyevaluated in a single run, multiple subsets are evaluated in a singlerun. Thus, in a single-pass and in real-time a sample is evaluated formultiple analytes. Measured F_(m) values for all beads assayed andclassified as belonging to a given subset may be averaged or otherwisemanipulated statistically to give a single meaningful data point,displayed in histogram format to provide information about thedistribution of F_(m) values within the subset, or analyzed as afunction of time to provide information about the rate of a reactioninvolving that analyte.

[0028] In a preferred embodiment, the beads will have two or morefluorochromes incorporated within or on them so that each of the beadsin a given subset will possess at least four different classificationparameters, e.g., C₁, C₂, C₃, and C₄. For example, the beads may be madeto contain a red fluorochrome (C₃), such as nile red, and bear an orangefluorochrome (C₄), such as Cy3 or phycoerythrin. A third fluorochrome,such as fluorescein, may be used as a source of the C_(n) or F_(m)signal. As those of skill in the art will recognize, additionalfluorochromes may be used to generate additional C_(n) signals. That is,given suitable fluorochromes and equipment, those of skill in the artmay use multiple fluorochromes to measure a variety of C_(n) or F_(m)values, thus expanding the multiplexing power of the system evenfurther.

[0029] In certain applications designed for more quantitative analysisof analyte concentrations or for kinetic studies, multiple subsets ofbeads may be coupled to the same reactant but at varying concentrationsso as to produce subsets of beads varying in density of bound reactantrather than in the type of reactant. In such an embodiment, the reactantassociated with classification parameter C₄, for example, may beincorporated directly into the reactive reagent that is coupled to thebeads, thereby allowing C₄ conveniently to serve as an indicator ofdensity of reactant on the bead surface as well as an indicator ofreactant identity.

[0030] To prepare subsets varying in reactant density one may, forexample, select, isolate, or prepare a starting panel of differentsubsets of beads, each subset differing from the other subsets in one ormore of C₁, C₂, or C₃. Each of those subsets may be further subdividedinto a number of aliquots. Beads in each aliquot may be coupled with areactant of choice that has been fluorescently labeled with afluorochrome associated with C₄ (e.g., Analyte A labeled with Cy3) underconditions such that the concentration or density of reactant bound tothe beads of each aliquot will differ from that of each other aliquot inthe subset. Alternatively, an entire subset may be treated with the C₄fluorochrome under conditions that produce a heterogeneous distributionof C₄ reactant on beads within the subset. The subset may then be sortedwith a cell sorter on the basis of the intensity of C₄ to yield furthersubsets that differ from one another in C₄ intensity.

[0031] One limitation of the alternative embodiment of using C₄ labeledreactant as a classification agent is that one must design the system sothat the value of C₄ as a classification parameter is not lost.Therefore, one must take care to assure that the C₄ intensities of allsubsets carrying reagent A differs from the C₄ intensities of allsubsets carrying reagents B, C, and so forth. Otherwise, C₄ would not beuseful as a parameter to discriminate reactant A from reactant B, etc.

[0032] With either embodiment, the number of subsets that can beprepared and used in practice of the invention is theoretically quitehigh, but in practice will depend, inter alia, on the level ofhomogeneity within a subset and the precision of the measurements thatare obtained with a flow cytometer. The intra-subset heterogeneity for agiven parameter, e.g., forward angle light scatter C₁, correlatesinversely with the number of different subsets for that parameter thatcan be discriminated by flow cytometric assay. It is therefore desirableto prepare subsets so that the coefficients of variation for the valueof each classification parameter (C₁, C₂, C₃, and C₄) to be used in agiven analysis is minimized. Doing this will maximize the number ofsubsets that can be discriminated by the flow cytometer. Bead subsetsmay be subjected to flow cytometric sorting or other procedures atvarious different points in preparation or maintenance of the beadsubsets to increase homogeneity within the subset. Of course, withsimple assays designed to detect only a few different analytes, moreheterogeneity can be allowed within a subset without compromising thereliability of the assay.

[0033] In an illustrative embodiment set forth here to explain onemanner in which the invention can work in practice, the beads are usedto test for a variety of antibodies in a fluid sample. A panel of beadsubsets having known varying C₁, C₂, C₃, and C₄ values is first preparedor otherwise obtained. The beads within each subset are then coupled toa given antigen of interest. Each subset receives a different antigen.The subsets are then pooled to form an assay beadset and may be storedfor later use and/or sold as a commercial test kit.

[0034] In the assay procedure, the beads are mixed with the fluid to beanalyzed for antibodies reactive with the variety of antigens carried onthe beads under conditions that will permit antigen-antibodyinteraction. The beads are labeled with a “secondary” reagent that bindsto antibodies bound to the antigens on the beads and that also bears themeasurement fluorochrome associated with parameter F_(m) (e.g.,fluorescein). A fluoresceinated antibody specific for immunoglobulin maybe used for this purpose. The beads are then run through a flowcytometer, and each bead is classified by its characteristicclassification parameters as belonging to subset-1, subset-2, etc. Atthe same time, the presence of antibodies specific for antigen A, B,etc., can be detected by measuring green fluorescence, F_(m), of eachbead. The classification parameters C₁, C₂, C₃, and C₄ allow one todetermine the subset to which a bead belongs, which serves as anidentifier for the antigen carried on the bead. The F_(m) value of thebead indicates the extent to which the antibody reactive with thatantigen is present in the sample.

[0035] Although assays for antibodies were used above as anillustration, those of ordinary skill in the art will recognize that theinvention is not so limited in scope, but is widely applicable todetecting any of a number of analytes in a sample of interest. Forexample, the methods described here may be used to detect enzymes or DNAor virtually any analyte detectable by virtue of a given physical orchemical reaction. A number of suitable assay procedures for detectionand quantification of enzymes and DNA (particularly as the result of aPCR process) are described in more detail below.

[0036] The present invention also provides a significant advance in theart by providing a rapid and sensitive flow cytometric assay foranalysis of genetic sequences that is widely applicable to detection ofRNA, differing alleles, and any of a number of genetic abnormalities. Ingeneral, the methods of the present invention employ a competitivehybridization assay using DNA coupled microspheresand fluorescent DNAprobes. Probes and microsphere-linked oligonucleotides could alsoinclude RNA, PNA, and non-natural nucleotide analogs.

[0037] In practice of the invention, oligonucleotides from a region of agene of interest, often a polymorphic allele or a region to which adisease associated mutation has been mapped, are synthesized and coupledto a microsphere (bead) by standard techniques such as by carbodiimidecoupling. A fluorescent oligonucleotide, complementary to theoligonucleotide on the bead, is also synthesized. To perform a test inaccordance with the invention, DNA which is to be tested is purified andeither assayed unamplified, or subjected to amplification by PCR,RT-PCR, or LCR amplification using standard techniques and PCRinitiation probes directed to amplify the particular region of DNA ofinterest. The PCR product is then incubated with the beads underconditions sufficient to allow hybridization between the amplified DNAand the oligonucleotides present on the beads. A fluorescent DNA probethat is complementary to the oligonucleotide coupled to the beads isalso added under competitive hybridization conditions. Aliquots of thebeads so reacted are then run through a flow cytometer and the intensityof fluorescence on each bead is measured to detect the level offluorescence which indicates the presence or absence of given sequencesin the samples.

[0038] For example, when beads labeled with an oligonucleotide probecorresponding to a non-mutated (wild-type) DNA segment are hybridizedwith the PCR product from an individual who has a non-mutated wild-typeDNA sequence in the genetic region of interest, the PCR product willeffect a significant competitive displacement of fluorescentoligonucleotide probe from the beads and, therefore, cause a measurabledecrease in fluorescence of the beads, e.g., as compared to a controlreaction that did not receive PCR reaction product. If, on the otherhand, a PCR product from an individual having a mutation in the regionof interest is incubated with the beads bearing the wild-type probe, asignificantly lesser degree of displacement and resulting decrease inintensity of fluorescence on the beads will be observed because themutated PCR product will be a less effective competitor for binding tothe oligonucleotide coupled to the bead than the perfectly complementaryfluorescent wild-type probe. Alternatively, the beads may be coupled toan oligonucleotide corresponding to a mutation known to be associatedwith a particular disease and similar principles applied. In themultiplexed analysis of nucleic acid sequences, bead subsets areprepared with all known, or possible, variants of the sequence ofinterest and then mixed to form a bead set. The reactivity of the testsample, e.g. PCR product, with the wild-type sequence and other variantscan then be assayed simultaneously. The relative reactivity of the PCRproduct with subsets bearing the wild-type or variant sequencesidentifies the sequence of the PCR product. The matrix of informationderived from this type of competitive hybridization in which the testsequence and the entire panel of probe sequences react simultaneouslyallows identification of the PCR product as wild-type, known mutant, orunknown mutant. The invention thus provides one with the ability tomeasure any of a number of genetic variations including point mutations,insertions, deletions, inversions, and alleles in a simple, exquisitelysensitive, and efficient format.

[0039]FIG. 1 is a block diagram of an illustrative hardware system forperforming a multiplex assay method in accordance with the invention.

[0040]FIG. 2 is a block diagram of an illustrative software system forperforming a multiplex assay method in accordance with the invention.

[0041]FIG. 3 is a flow-chart for a preprocessing phase in accordancewith the inventive multiplexed assay method.

[0042]FIG. 4 shows an assay database in accordance with the invention.

[0043]FIG. 5 shows a baseline data acquisition table for an illustrativemultiple analyte assay in accordance with the invention.

[0044]FIG. 6 shows an assay definition table in accordance with theinvention.

[0045]FIG. 7 shows a discriminant table for an illustrative multipleanalyte assay in accordance with the invention.

[0046]FIG. 8 shows a decision tree view of the illustrative discriminantfunction table of FIG. 7.

[0047]FIG. 9 is a flow-chart for a real-time analysis phase of amultiple analyte assay in accordance with the invention.

[0048]FIG. 10 shows a results table for an illustrative multiple analyteassay in accordance with the invention.

[0049]FIG. 11 shows a interpretation table for an illustrative multipleanalyte assay in accordance with the invention.

[0050]FIG. 12 is a flow-chart for an interpretation phase of a multipleanalyte assay in accordance with the invention

[0051]FIGS. 13a through 13 e show an assay database in accordance withthe invention for a specific experimental example.

[0052]FIG. 14 shows a decision tree view for an illustrative(experimental example) discriminant table.

[0053]FIGS. 15a, 15 b, and 15 c show individual inhibition assays forIgG, IgA, and IgM antibodies.

[0054]FIGS. 16a, 16 b, and 16 c show cross reactivity determinationsbetween IgG, IgA, and IgM assay components.

[0055]FIG. 17 shows the determination of human IgG concentrations byflow cytometry.

[0056]FIG. 18 shows the determination of human IgA concentrations byflow cytometry.

[0057]FIG. 19 shows the determination of human IgM concentrations byflow cytometry.

[0058]FIG. 20 shows the simultaneous determination of human IgG, IgA,and IgM concentrations by flow cytometry.

[0059]FIG. 21 shows the specificity of monoclonal antibody MAB384binding towards bead immobilized epitope sequences.

[0060]FIG. 22 shows the specificity of monoclonal antibody MAB384binding in the presence of soluble epitope containing peptide.

[0061]FIG. 23 shows the specificity of monoclonal antibody MAB384binding in the presence of soluble biotin.

[0062]FIG. 24 shows the detection of anti-Rubella IgG antibodies by asandwich assay between rubella coated beads and a fluorescent goatanti-human IgG antibody.

[0063]FIG. 25 shows a calibration assay using serial dilutions ofanti-Rubella IgG antibodies in a sandwich assay between rubella coatedbeads and a fluorescent goat anti-human IgG antibody.

[0064]FIGS. 26a and 26 b show the simultaneous assay for six anti-ToRCHIgG, and simultaneous assay for the six anti-ToRCH IgM antibodies.

[0065]FIG. 27 shows the determination of IgG anti-grass allergenactivities for six dogs.

[0066]FIG. 28 shows the determination of IgE anti-grass allergenactivities for six dogs.

[0067]FIG. 29 shows the multiple analyte IgG and IgE screening of dogserum A96324 for sixteen grass allergens

[0068]FIG. 30 shows the multiple analyte IgG and IgE screening of dogserum A96325 for sixteen grass allergens

[0069]FIG. 31 shows the multiple analyte IgG and IgE screening of dogserum A96319 for sixteen grass allergens

[0070]FIG. 32 shows the multiple analyte IgG and IgE screening of dogserum A96317 for sixteen grass allergens

[0071]FIG. 33 shows the multiple analyte IgG and IgE screening of dogserum A96326 for sixteen grass allergens

[0072]FIG. 34 shows the multiple analyte IgG and IgE screening of dogserum A96323 for sixteen grass allergens

[0073]FIG. 35 shows an antibody pair analysis for use with a humanchorionic gonadotropin capture assay.

[0074]FIG. 36 shows the use of bead linked antibody MAB602 withfluorescently labeled antibody AB633 in a human chorionic gonadotropincapture assay.

[0075]FIG. 37a and 37 b show cross reactivity analyses betweencomponents of an anti-hCG capture system and an anti-AFP capture system.

[0076]FIGS. 38a and 38 b compare the effects of eliminating wash stepsin hCG and AFP capture system assays.

[0077]FIGS. 39a and 39 b show the determination of hCG and AFPconcentrations in samples and standards using a homogeneous captureassay format.

[0078]FIG. 40 shows the inhibition of Anti-IgG binding to bead based IgGby soluble IgG antibodies. Inhibition was determined at fiveconcentrations of soluble IgG, and four IgG loading levels on the beads.

[0079]FIG. 41 shows the slope of the inhibition pattern across the fourloading levels of IgG on the beads plotted against the soluble IgGconcentration.

[0080]FIG. 42 shows a five point standard curve derived from inhibitiondata of the 50 μg/mL IgG bead set.

[0081]FIGS. 43a through 43 c show DNA detection using a double strandedcompetitor and a wild-type “B” oligonucleotide probe.

[0082]FIGS. 44a and 44 b show DNA detection using a single strandedcompetitor and a wild-type “B” oligonucleotide probe.

[0083]FIG. 45 shows the differentiation by orange and red fluorescenceof fourteen bead sets.

[0084]FIG. 46 shows a titration of a fluorescent oligonucleotide in thepresence or absence of an inhibitor. Beads bearing complementaryoligonucleotides were used in a capture assay.

[0085]FIG. 47 shows the inhibition of binding between a fluorescentoligonucleotide and its complementary oligonucleotide bound to a bead.Varying concentrations of complementary and point mutant competitorswere used in the determination.

[0086]FIG. 48 shows the efficacy of inhibitors across fourteen DNAsequence binding sets.

[0087]FIG. 49 shows the typing of four simulated alleles of DQA1.

[0088]FIG. 50 shows the typing of five known, homozygous DQA1 alleles.

[0089]FIGS. 51 a through 51f show the results of an exemplarymultiplexed assay according to the invention.

[0090] According to the present invention, assay components and methodsfor the measurement of enzymes, DNA fragments, antibodies, and otherbiomolecules are provided. The inventive technology improves the speedand sensitivity of flow cytometric analysis while reducing the cost ofperforming diagnostic and genetic assays. Further, and of tremendoussignificance, a multiplexed assay in accordance with the inventionenables the simultaneous automated assay of multiple (at least an orderof magnitude greater than available in the prior techniques)biomolecules or DNA sequences in real-time.

[0091] As those of ordinary skill in the art will recognize, theinvention has an enormous number of applications in diagnostic assaytechniques. Beadsets may be prepared, for example, so as to detect orscreen for any of a number of sample characteristics, pathologicalconditions, or reactants in fluids. Beadsets may be designed, forexample, to detect antigens or antibodies associated with any of anumber of infectious agents including (without limitation, bacteria,viruses, fungi, mycoplasma, rickettsia, chlamydia, and protozoa), toassay for autoantibodies associated with autoimmune disease, to assayfor agents of sexually transmitted disease, or to assay for analytesassociated with pulmonary disorders, gastrointestinal disorders,cardiovascular disorders, and the like. Similarly, the beadset may bedesigned to detect any of a number of substances of abuse, environmentalsubstances, or substances of veterinary importance. An advantage of theinvention is that it allows one to assemble a panel of tests that may berun on an individual suspected of having a syndrome to simultaneouslydetect a causative agent for the syndrome.

[0092] Suitable panels may include, for example, a tumor marker panelincluding antigens such as prostate-specific antigen (PSA),carcinoembryonic antigen (CEA), and other suitable tumor markers; aregional allergy panel including pollen and allergens tested for byallergists of a particular region and comprising allergens known tooccur in that region; a pregnancy panel comprising tests for humanchorionic gonadotropin, hepatitis B surface antigen, rubella virus,alpha fetoprotein, 3′ estradiol, and other substances of interest in apregnant individual; a hormone panel comprising tests for T4, TSH, andother hormones of interests; an autoimmune disease panel comprisingtests for rheumatoid factors and antinuclear antibodies and othermarkers associated with autoimmune disease; a blood borne virus paneland a therapeutic drug panel comprising tests for Cyclosporin, Digoxin,and other therapeutic drugs of interest.

[0093] Bead Technology

[0094] An important feature of the flow cytometric technology andtechniques described here is the fabrication and use of particles (e.g.,microspheres or beads that make up a beadset). It is through the use ofappropriately labeled homogeneous bead subsets, combined to produce apooled beadset, that the instant multiplexed assay method is practiced.Beads suitable for use as a starting material in accordance with theinvention are generally known in the art and may be obtained frommanufacturers such as Spherotech (Libertyville, Ill.) and MolecularProbes (Eugene, Oreg.). Once a homogeneous subset of beads is obtained,the beads are labeled with an appropriate reactant such as abiomolecule, DNA sequence, and/or other reactant. Known methods toincorporate such labels include polymerization, dissolving, andattachment.

[0095] A Method for the Multiplexed Assay of Clinical Samples

[0096] Development of a multiplexed assay for use in accordance with theinvention can be divided into three phases: (1) preprocessing, (2)real-time analysis, and (3) interpretation. During the preprocessingphase, baseline data is collected independently, via flow cytometrictechniques, for each of an assay's bead subsets. Baseline data is usedto generate a set of functions that can classify any individual bead asbelonging to one of the assay's subsets or to a rejection class. Duringthe analysis phase, flow cytometric measurements are used to classify,in real-time, each bead within an exposed beadset according to theaforementioned functions. Additionally, measurements relating to eachsubset's analyte are accumulated. During the interpretation phase theassay's real-time numerical results are associated with textualexplanations and these textual explanations are displayed to a user.

[0097] The inventive method allows the detection of a plurality ofanalytes simultaneously during a single flow cytometric processing step.Benefits of the inventive multiplex assay method include increased speedand reduced cost to analyze a clinical sample.

[0098] System Hardware

[0099]FIG. 1 shows, in block diagram form, a system for implementing theinventive multiplexed assay method. Flow cytometer 100 output consistsof a series of electrical signals indicative of one or more specifiedmeasured characteristics on each bead processed. These measurementsignals are transmitted to computer 105 via data bus 110 and interfaceboard 115. During the preprocessing phase, the signals are used by thecomputer to generate an assay database. During the real-time analysisphase, the signals are processed by the computer (using the assaydatabase) in accordance with the inventive method to produce amultiplexed/simultaneous assay of a clinical sample.

[0100] Flow cytometer 100 operates in a conventional manner. That is,beads are processed by illuminating them, essentially one at a time,with a laser beam. Measurements of the scattered laser light areobtained for each illuminated bead by a plurality of optical detectors.In addition, if a bead contains at least one appropriate fluorescingcompound it will fluoresce when illuminated. A plurality of opticaldetectors within the flow cytometer measure fluorescence at a pluralityof wavelengths. Typical measured bead characteristics include, but arenot limited to, forward light scatter, side light scatter, redfluorescence, green fluorescence, and orange fluorescence. One ofordinary skill in the use of flow cytometric techniques will recognizethat the use of green fluorescent markers or labels can causecross-channel interference between optical detectors designed to detectgreen and orange wavelengths (e.g., approximately 530 nanometers andapproximately 585 nanometers respectively). A training set of beads, incombination with standard data manipulation, can correct for thiscross-channel interference by providing the physical measurementsrequired for mathematical correction of the fluorescence measurements.

[0101] One of ordinary skill will further recognize that manyalternative flow cytometer setups are possible. For instance, additionalcolor sensitive detectors could be used to measure the presence of otherfluorescence wavelengths. Further, two or more laser beams can be usedin combination to illuminate beads as they flow through the cytometer toallow excitation of fluorochromes at different wavelengths.

[0102] Computer 105 can be a conventional computer such as a personalcomputer or engineering workstation. In one embodiment, the computer isa personal computer having an Intel “486” processor, running MicrosoftCorporation's “WINDOWS” operating system, and a number of ISA expansionslots.

[0103] Interface board 115 is designed to plug into one of thecomputer's 100 ISA (Industry Standard Architecture) expansion slots.While the design of an interface board is, in general, different foreach specific type of flow cytometer 100, its primary functions include(1) receiving and parsing measurement data signals generated by the flowcytometer's detectors, (2) receiving control parameter statusinformation from the flow cytometer, and (3) sending control parametercommands to the flow cytometer. The precise manner in which thesefunctions are carried out are dependent upon the type (make and model)of the flow cytometer used. In one embodiment, employing aBecton-Dickinson “FACSCAN” flow cytometer (San Jose, Calif.), theinterface board uses control signals generated by the flow cytometer todistinguish measurement data and flow cytometer parameter and controlsignals. Measured data include forward light scatter, side lightscatter, red fluorescence, green fluorescence, and orange fluorescence.Parameter and control signals include flow cytometer amplifier gainadjustments and status information.

[0104] While the design of an interface board 115 for use with theinventive assay method would be a routine task for one skilled in theart of diagnostic medical equipment design having the benefit of thisdisclosure, an important aspect for any interface board is its abilityto accommodate the transmission data rate generated by whatever flowcytometer is used. For example, the “FACSCAN” flow cytometer cantransmit a 16-bit (2 byte) word every 4 microseconds resulting in burstdata rates of 500,000 bytes per second. Microfiche appendix A provides adetailed source code embodiment of the inventive assay method for usewith the “FACSCAN” flow cytometer.

[0105] Data bus 115 provides a physical communication link between theflow cytometer 100 and the interface board 110. Its physical andelectrical characteristics (e.g., data width and bandwidth) aredependent upon the capabilities of the flow cytometer. It is noted thatthe data bus need not be a totally digital bus. If the flow cytometerdoes not include analog-to-digital conversion of measured beadcharacteristics (e.g., light scatter and fluorescence signals), then thedata bus must communicate these analog signals to the interface board.It is then necessary that digital conversion of these signals beprovided by either the interface board, or another peripheral devicebefore the data is transmitted to the computer 105.

[0106] System Software

[0107] As shown in FIG. 2, the software architecture for the inventiveassay method can be divided into two parts. A graphical user interface(GUI) 200 provides the means by which a user (1) receives assay resultsand (2) interacts with the flow cytometer. A dynamically linked library(DLL) 205 provides the means through which the inventive real-time assayis performed and includes routines necessary to (1) interact withinterface board 115 and (2) send and receive information to the flowcytometer 100.

[0108] An important aspect of the inventive assay method is that itperforms a simultaneous analysis for multiple analytes in real-time. Oneof ordinary skill in the art of computer software development willrealize that real-time processing can impose severe time constraints onthe operational program code, i.e., the DLL 205. For example, the“FACSCAN” flow cytometer can process, or measure, approximately 2,000beads per second, where each bead is associated with eight 16-bit datavalues. Thus, to process flow cytometer data in real-time from a“FACSCAN,” the DLL should be able to accept, and process, at aconsistent data rate of at least 32,000 bytes per second. The need toaccommodate this data rate, while also having sufficient time to performreal-time analysis based on the data, will generally necessitate thatsome of the DLL code be written in assembly language.

[0109] In a current embodiment, the GUI 200 is implemented in the visualbasic programming language and the DLL 205 is implemented in C andassembly language programming. Microfiche appendix A contains sourcecode listings for one embodiment of the GUI and DLL.

[0110] Preprocessing

[0111] A function of the preprocessing phase is to generate an assaydatabase for use during the real-time analysis of an exposed beadset(clinical sample). Thus, preprocessing is performed prior to combiningseparately labeled bead subsets to form assay beadsets. Assaydefinition, discriminant function definition, and interpretation tablesare created at the time an assay beadset is created. FIG. 3 shows, inflow chart form, the steps taken during the preprocessing phase.

[0112] A bead subset is characterized by (1) the analyte it is designedto identify, (2) one or more classification parameters C₁ . . . C_(n),and (3) one or more measurement parameters F_(ml)-F_(mx). During thepreprocessing phase the classification parameters are used to generate aset of functions, referred to as discriminant functions, that canclassify a bead as belonging to one of the assay's subsets or arejection class. Measurement parameters are used during the real-timeanalysis phase to determine if a specified analyte is present in theclinical sample being analyzed.

[0113] The precise number of individual beads contained in any givensubset is relatively unimportant, the only significant criterion beingthat a sufficient number are used so that a good statisticalcharacterization of the subset's parameters can be achieved during thereal-time analysis phase. In a current embodiment, each bead subsetcontains an equal number of beads. One of ordinary skill in the fieldwill recognize that the precise number of beads within any given beadsubset can vary depending upon many factors including, but not limitedto, the number of analytes an assay beadset is designed to detect, theuniformity of the labeled beads (with respect to each of the measuredparameters C₁. . . C_(n), F_(ml) . . . F_(mx)), and the penalty ofmisclassifying (e.g., making a type 1 or type 2 classification error) abead during analysis.

[0114] During preprocessing, each bead in an unexposed subset ismeasured by a flow cytometer 100 and the resulting data valuesaccumulated for later use 300. For example, if the flow cytometermeasures n classification parameters and x measurement parameters, i.e.,generates (n+x) values for each bead, data for each of the subset's(n+x) parameters are updated based on each bead's measurements. Thisdata collection step is repeated independently for each subset in theassay's beadset 305. The collection of such data for each of an assay'ssubsets constitutes an assay's baseline data.

[0115] After an assay's baseline data has been collected, a set ofdiscriminant functions are determined 310. During real-time analysis,the discriminant functions are used to classify a bead into one of theassay's bead subsets or a rejection class based solely on the measuredclassification parameters, C₁ . . . C_(n). This step, in principle andpractice, is a problem of multi-dimensional classification or clusteranalysis. Many prior art techniques and commercial software programsexist to perform this task.

[0116] Beads are generally manufactured in large quantities referred toas batches. Each bead in a batch is of nearly identical size and hassubstantially the same dye absorption capacity. In light of thismanufacturing process, bead subsets can be created using precisedilutions of chosen dyes and, because of their nearly identical size,all classification parameters will exhibit essentially equal variances.By correcting for scaling of the photo-multipliers within a flowcytometer, a linear classification rule can be generated. Further, sincethere are equal quantities of beads in each subset, the priorprobabilities will be equal. This allows use of Fisher's lineardiscriminant technique to calculate the discriminant functions whichdefine classification boundaries. See, Fisher, “The Use of MultipleMeasurements in Taxonomic Problems,” Annals of Eugenics, 7, 179-188(1936). For instance, linear hierarchical discriminant functions may bechosen which are equidistant, in a Euclidean sense, between the centersor centroids of any two of an assay's bead subsets. Notwithstanding thepresent example, other types of discriminant functions, such asquadratic functions and those discriminating on more than twoclassification parameters at once, are also possible.

[0117] In addition to the discriminant functions, a set of thresholdvalues are chosen which are used during the real-time analysis phase todetect the presence of a target analyte. For example, assume measurementparameter F_(ml) is used to detect analyte-A. During preprocessing, thebaseline or unexposed value for F_(ml) is measured and accumulated forthat subset's beads. Analyte-A's threshold could then, for example, beset to F_(ml)'s baseline mean value plus one standard deviation ofF_(ml)'s baseline value. One of ordinary skill will recognize that theprecise function or value selected for a threshold depends upon theparameter being measured (e.g., its distribution) and the cost of makinga classification error (e.g., a type 1 or a type 2 error). It is routinethat such values be based on an empirical review of the baseline data.The important criterion is that the threshold reliably distinguishbetween the presence and absence of the target analyte in an exposedassay beadset.

[0118] After baseline data for each of an assay's bead subsets arecollected and discriminant functions and analyte threshold values areestablished, an assay database is generated 315.

[0119] Assay Database

[0120] As shown in FIG. 4, an assay database 400 consists of an assaydefinition table 405, a discriminant function table 410, a results table415, and an interpretation table 420. See FIG. 4.

[0121] The assay definition table 405 defines an assay which, asdescribed above, comprises two or more bead subsets each of which isdesigned to detect a specified analyte. Each row in the assay definitiontable describes a bead subset and contains the following entries: (1)assay name, (2) subset name, (3) subset token, (4) baseline values foreach of the subset's measurement parameters F_(ml)-F_(mx), and (5)test-type token. The subset name entry is a text string identifying thesubset by, for example, the type of analyte it is labeled to detect. Thesubset token is a unique subset identifier. The measurement parameterbaseline entries are used during the interpretation phase to associate anumerical result (collected during the real-time analysis of a clinicalsample) with a textual output string. Finally, the test-type tokenidentifies which one of a possible plurality of interpretation tests toperform on the collected (real-time) data during the interpretationphase.

[0122] The discriminant function table 410 is used to systematically setforth an assay's set of discriminant functions. Each row in thediscriminant function table implements a single discriminant functionand includes entries for (1) the assay's name, (2) a unique rowidentifier, (3) one or more classification parameters upon which toevaluate, (4) high and low discriminant values for each of the listedclassification parameters, and (5) evaluation tokens which are assignedas a result of evaluating the discriminant function.

[0123] The results table 415 is used to store, or accumulate, data on anassay's beadset during the real-time analysis phase of the inventivemethod and is discussed further in Section 6.2(d).

[0124] The interpretation table 420 provides a means to associate textmessages with each enumerated assay result and is discussed further inSection 6.2(e).

[0125] Preprocessing Example

[0126] Consider an assay beadset designed to simultaneously detect fouranalytes: analyte-A, analyte-B, analyte-C, and analyte-D. Thus, theassay's beadset is comprised of four bead subsets, each labeled for adifferent analyte. Suppose further that the assay beadset is to beprocessed by a Becton-Dickinson Immunocytometry Systems “FACSCAN” flowcytometer, For each bead processed, the “FACSCAN” measures forward lightscatter, side light scatter, red fluorescence, orange fluorescence, andgreen fluorescence. Let classification parameter C₁ be forward lightscatter, classification parameter C₂ be side light scatter,classification parameter C₃ be red fluorescence, classificationparameter C₄ be orange fluorescence, and measurement parameter F_(ml) begreen fluorescence. (This notation implies that each bead in a subset islabeled with a green fluorophore bearing, for example, an antibody ordye specifically targeted to that subset's analyte.)

[0127] After preparing each of the four subsets and before they arecombined to form the assay beadset, they are processed by the flowcytometer and their measured data are accumulated: values for each ofthe parameters C₁, C₂, C₃, C₄, and F_(ml) are recorded for each bead.Each bead subset is similarly processed. Completion of this taskconstitutes completion of baseline data acquisition.

[0128] Using baseline data, the assay's beads are clustered in thefour-dimensional parameter space defined by C₁, C₂, C₃, and C₄. Theresult of this cluster analysis is that each subset is characterized bya mean (μ) and standard deviation (C) for each of its fourclassification parameters. See FIG. 5. As previously noted, the precisenumber of individual beads contained in any given bead subset can becalculated by those of ordinary skill in the art. This calculation isrequired to obtain good statistical characterization of the subset'sparameters—e.g., small, or relatively fixed, coefficient of variationsfor each parameter.

[0129] As shown in FIG. 6, the assay definition table 405 is comprisedof general information relevant to the overall diagnostic function ofthe assay. For instance, in a genotyping assay, each of the assay'ssubset's may be assigned a token used for identification: e.g., token 46represents the bead subset labeled to detect a wildtype coding sequencefor a specified gene; subset tokens 21, 50, and 5 represent subsetslabeled to detect various mutant type coding sequences for a specifiedgene(s). Additionally, measurement parameter F_(ml)'s baseline (in thisexample the mean) and standard deviation values are listed. Finally, atest-type token is listed. In the current embodiment a test-type tokenof ‘0’ means an OVER/UNDER interpretation test is to be performed and atest-type token of ‘1’ means a SHIFT interpretation test is to beperformed. See Section 6.2(f) for further discussion of these issues.

[0130] Discriminate functions are generated by viewing the assay'sbaseline data graphically in three dimensions and creating planes toseparate the different subset clusters. These “planes” are created byapplying Fischer's Linear Discriminant to the n-dimensionalclassification parameter space. A populated discriminate function tablebased on the baseline data of FIG. 5 is shown in FIG. 7.

[0131] The discriminant function table provides a systematic means ofevaluating a series of classification values (C₁, C₂, C₃, C₄) in orderto classify a bead. In general bead classification proceeds by enteringthe discriminant function table at row 0, performing a test on aspecified parameter (e.g., C₁, C₂, C₃, or C₄) and then, depending uponthe result, either classifying the bead or proceeding to another testwhich involves evaluating a different row in the table. For example,suppose bead A has the following measured classification parametervalues: C₁=V₁, C₂=V₂, C₃=V₃, and C₄=V₄. Classification of bead A via thediscriminant function table of FIG. 7 begins as follows (the pseudo-codebelow would demonstrate to those skilled in the art of programming thelogic involved in the classification process):

[0132] 1. Enter table at row 0 with measured values for C₁, C₂, C₃, andC₄.

[0133] 2. If (LOW VALUE=500)≦(PARAMETER=C₁=V₁)≦(HIGH VALUE=620) then(result=TRUE), else (result=FALSE).

[0134] 3. If (result=TRUE) and (TRUE ROW ID≈0), then re-enter table atTRUE ROW ID, else

[0135] 4. If (result=TRUE) and (TRUE ROW ID=0), then (class=TRUE TOKEN).

[0136] 5. If (result=FALSE) and (FALSE ROW ID≠0), then re-enter table at(row=FALSE ROW ID), else

[0137] 6. If (result=FALSE) and (FALSE ROW ID=0), then (class=FALSETOKEN).

[0138] 7. If (TRUE TOKEN or FALSE TOKEN)=0, then (class=reject class).

[0139] One of ordinary skill will recognize from the above discussionthat a discriminant function table embodies a (classification) decisiontree. FIG. 8 shows this relationship for the discriminant function tableof FIG. 7 explicitly. A discussion of the discriminant function table asit relates to the real-time processing of an exposed assay beadset isprovided in Section 6.2(d). Once a beadset is preprocessed, the data maybe employed in real-time analysis of many assays using that set. One ofordinary skill in the art will also recognize that instead of a decisiontree, a bitmap or look up table could be used to classify the bead sets.

[0140] Real-Time Analysis

[0141] Once a collection of bead subsets have been characterized asdescribed above and combined to form an assay beadset, the beadset maybe exposed to a test sample. That is, they may be used to analyze aclinical sample. After exposure the beadset is ready for real-timeanalysis. The real-time analysis phase is initiated by installing theexposed beads into a conventional flow cytometer for processing.

[0142] As described above, for each bead processed a flow cytometer 100generates electrical signals indicative of a plurality of measuredparameters, C₁ . . . C_(n), F_(ml) . . . F_(mx). These values aretransmitted to computer 105 via data bus 110 and interface board 115.Values for a bead's classification parameters C₁ . . . C_(n) are used toevaluate the assay's discriminant functions, as encoded in adiscriminant function table 410, the result of which is an initialclassification of the bead into one of the assay's bead subsets or areject class.

[0143] After this initial classification, a bead's measuredclassification parameter values C₁ . . . C_(n) can be checked againsttheir (C₁ . . . C_(n)) baseline values to determine if it is“reasonable” to classify the bead as belonging to the initiallyidentified class. In a current embodiment, this reasonableness test isimplemented by computing the distance between the measuredclassification parameter values and the mean values obtained duringpreprocessing. If the measured values for C₁ . . . C_(n) for aparticular bead are sufficiently distant from the identified subsetsbaseline values, the bead is assigned to a reject class. Use of thistechnique allows for the rejection of beads that were initiallymisclassified and improves the overall reliability of the analysis.

[0144] To ensure proper classification, a preferred embodiment's pooledbeadset will include a bead subset which has no bound reactants (e.g., aplacebo bead subset) in a known ratio to the beadset's other subsets.

[0145] It is noted that when a beadset is comprised of beadsmanufactured in a single batch, the above described reasonableness testcan be incorporated into the linear discriminant functions by creatingreject space between all subsets. However, when a beadset is comprisedof beads from more than one batch a Euclidean (or similar) distancemeasure is needed to validate the classification result.

[0146] Once a bead is assigned its final classification, the assay'sresults table 415 is updated to reflect the newly classified bead'smeasurement parameter values F_(ml) . . . F_(mx). This data acquisition,classification, and update process is repeated for each bead in theassay beadset in real-time. FIG. 9 shows, in block diagram form, thegeneral steps performed during the real-time analysis phase of a methodin accordance with the invention.

[0147] In one embodiment the following data are accumulated in theresults table for each class (subset) of bead in the assay: (1) totalcount of the number of beads detected in the specified class, (2) arunning sum for each measurement parameter F_(ml)-F_(mx), (3) for eachmeasurement parameter the total count of the number of beads in theclass whose measurement value is less than the parameter's baselinevalue, and (4) for each measurement parameter the total count of thenumber of beads in the class whose measurement value is more than theparameter's baseline value.

[0148] Real-Time Analysis Example

[0149] In the illustrative embodiment introduced in Section 6.2(c), theassay beadset is designed to simultaneously detect four analytes usingfour classification parameters (C₁ represents forward light scatter, C₂represents side light scatter, C₃ represents red fluorescence, and C₄represents orange fluorescence) and one measurement parameter (F_(ml)representing green fluorescence). After exposing the beadset to asuitable biological sample, it is placed into a flow cytometer 100 whichprocesses each bead (e.g., measures parameters C₁, C₂, C₃, C₄, andF_(ml)) and transmits to computer 105 signals indicative of thesemeasurements via data bus 110 and interface board 115.

[0150] For each bead processed by the flow cytometer, values for C₁, C₂,C₃, and C₄ are evaluated in accordance with the discriminant functiontable shown in FIG. 7 to initially classify the bead as belonging to aparticular subset, for example, in a genetic analysis intended to detectmutations in the Kras oncogene, the classification could proceed asfollows: (1) class 46, Kras CODON 46 WILDTYPE, (2) class 21, Kras CODON21 MUTANT, (3) class 50, Kras CODON 50 MUTANT, (4) class 5, Kras CODON 5MUTANT, or (5) a reject class. (See FIG. 8 for a decision treerepresentation of the discriminate function table of FIG. 7.) If thebead is initially classified as belonging to any class except the rejectclass, a reasonableness test is performed on the bead's classificationparameter values, C₁-C_(n). For example, if the bead received an initialclassification of class 50 and its C₁ value is more than two standarddeviations away from its mean, the bead is given a final classificationof reject. Otherwise the bead's final classification is the same as itsinitial classification—50.

[0151] If the bead's final classification is other than reject, itsF_(ml) value is used to update the assay's results table in thefollowing manner (see FIG. 10):

[0152] 1. Identifying, based on the bead's classification token (i.e.,subset token 46, 21, 50, or 5), the row in the results table which is tobe updated.

[0153] 2. Incrementing the identified row's COUNT value. The COUNT valuereflects the total number of beads of the specified class that have beenidentified during the analysis.

[0154] 3. Adding the bead's F_(ml) value to the value contained in therow's SUM column. The SUM value reflects a running sum of the identifiedclasses measurement values.

[0155] 4. If the bead's F_(ml) value is greater than F_(ml)'s base value(determined during the preprocessing phase, see FIG. 6), thenincrementing the row's OVER COUNT value. The OVER COUNT value reflectsthe total number of beads of the specified class that have beenprocessed whose F_(ml) values are above that of baseline.

[0156] 5. If the bead's F_(ml) value is less than F_(ml)'s base value(as determined during the preprocessing phase, see FIG. 6), thenincrementing the row's UNDER COUNT value. The UNDER COUNT value reflectsthe total number of beads of the specified class that have beenprocessed whose F_(ml) values are below that of baseline.

[0157] In a preferred embodiment, data (i.e., count, and measured F_(ml)values) for each bead classified as a reject can also be collected.

[0158] Interpretation

[0159] Following the real-time classification and accumulation ofresults as described above, the user may select to see a text basedpresentation or interpretation of the assay's numerical results. Duringthe interpretation phase the assay's real-time numerical results areassociated with textual explanations. These textual explanations can bedisplayed to the user.

[0160] It is the function of the interpretation table 420 to associatetextual descriptions of an assay's possible outcomes with an actualassay's numerical results. Each row in the interpretation table providesthe necessary information to make a single interpretation and typicallyincludes entries for (1) the assay's name, (2) a subset tokenidentifying the class or subset on which the interpretation is based,(3) an outcome identifier for the identified subset, (4) a test-typetoken, (5) high and low discriminant values for each measurementparameter utilized in the identified test, and (6) a text stringdescribing the row's result.

[0161] The test-type token identifies which one of a possible pluralityof interpretation tests to perform on the collected (real-time) dataduring the interpretation phase. In a current embodiment the test-typetoken is either ‘0’ or ‘1’. A value of ‘0’ indicates an OVER/UNDERinterpretation test is to be performed. A value of ‘1’ indicates a SHIFTinterpretation test is to be performed. These tests are defined in thefollowing manner: $\begin{matrix}{{{{OVER}\text{/}{UNDER}\quad {Test}\quad {Value}} = \frac{{OVER}\quad {COUNT}}{{UNDER}\quad {COUNT}}},{and}} \\{{{{SHIFT}\quad {Test}\quad {Value}} = \frac{\frac{SUM}{COUNT}}{{Baseline}\quad F_{m}\quad {Value}}},}\end{matrix}$

[0162] where the variables OVER COUNT, UNDER COUNT, SUM, COUNT, andbaseline F_(m) are described above in Section 6.2(d).

[0163] The OVER/UNDER test is generally used for qualitativemeasurements where the level of reactivity of beads is an indication ofthe condition or concentration of a biomolecule present in the sample.The shift test is used where the result sought is a determination of thea minimally detectable level of a particular biomolecule. One ofordinary skill will recognize that many other tests could be performed.Examples include ranking, stratification, ratio of means to a standard,or to each other, etc.

[0164] In general an interpretation table 420 may associate any numberof entries or interpretations (e.g., rows within the table) with asingle assay class or bead subset. For instance, bead subset Y couldhave a single measurement parameter (F_(ml)) associated with it and thismeasurement parameter could indicate, depending upon its value, that oneor more interpretations are appropriate.

[0165] Note, the contents of the interpretation table 420 are generatedduring the preprocessing phase. This implies that the target assay beunderstood and that the various assay results be considered prior toconstruction of multiplexed assays.

[0166] Interpretation Example

[0167] Consider again the assay beadset, introduced above, designed tosimultaneously detect four analytes. FIG. 11 shows a sampleinterpretation table for this assay. Interpretation of the assay'sreal-time numerical results is initiated by, for example, the userselecting “interpret results” via the inventive method's graphical userinterface.

[0168] As described above, each bead subset (class) within an assay hasan entry or row in the results table, FIG. 10. The general procedure forinterpreting an assay's real-time numerical results is shown inflow-chart form in FIG. 12. In general, each row of the results table ismatched against every row in the interpretation table with the samesubset token. If the result of performing the specified test is betweenthe identified row's low and high values, then the associated textualmessage is displayed to the user. When all rows in the interpretationtable for a single results table row have been checked, the next resultstable row is evaluated. This process is repeated until the every row inthe interpretation table has been compared to the appropriate resultstable entry.

[0169] As a specific example, consider the interpretation of subset 50's(KRAS CODON 50 MUTANT, see FIG. 6) results table entry. The subset'stoken, 50, is used to identify three rows in the interpretation table(having outcome IDs of 1, 2, and 3) that contain information regardingevaluation of the mutant analyte. For the first identified row, thetest-type token indicates a SHIFT type interpretation test is to beperformed. Performing this test, as defined above, yields:${{SHIFT}\quad {Test}\quad {Value}} = {\frac{\frac{SUM}{COUNT}}{{Baseline}\quad F_{m}\quad {Value}} = {\frac{\frac{1,700,000}{1,000}}{170} = 10}}$

[0170] Next, the computed SHIFT test value is compared against eachinterval in the identified rows of the interpretation table. For the rowhaving OUTCOME ID equal to 1, since (LOW VALUE=10)≦SHIFT TestValue=10≦(HIGH VALUE=667) is true, that row's INTERPRETATIONentry—“identical complementary strand”—is displayed to the user. Thisprocess is repeated for subset 50's remaining two rows in theinterpretation table. Further, this process is repeated for each row inthe results table.

[0171] The result of the interpretation phase is a series of textualmessages that describe the results of the assay. Conclusion of theinterpretation phase marks the end of the assay.

[0172] Operational Considerations

[0173] Assay definition, discriminant function definition, andinterpretation tables are created at the time an assay beadset iscreated. Baseline classification data is collected only once for a givenassay. That is, once an assay is defined and its baseline data isobtained, any number of beadsets can be manufactured to perform theanalysis. To allow this “sharing” of baseline data the assay beadset maycontain a center or calibration bead subset.

[0174] As would be known to those of ordinary skill in the field, acalibration beadset can be used to adjust any given flow cytometer to astandard. Calibration beadsets are typically processed separately froman assay. Further, calibration is generally performed daily. The purposeof calibration is to adjust the sensitivity of a flow cytometer'sphotomultipliers to accommodate day to day and machine to machinedifferences.

[0175] Unlike prior art calibration techniques which are performedmanually, the processing of a calibration beadset and the adjustment offlow cytometer operational parameters (e.g., photomultiplier voltages)is performed under software control automatically. See microficheappendix A for embodiment details.

[0176] Antibody Detection

[0177] Assays for antibody are widely used in medicine and clinicalanalysis for an wide variety of purposes, from detection of infectionsto determination of autoantibody. The following example illustrates useof the inventive method in an antibody assay and assumes the use of aflow cytometer capable of providing at least five measurements for eachbead processed: forward light scatter as classification parameter C₁,side light scatter as classification parameter C₂, red fluorescence asclassification parameter C₃, orange fluorescence as classificationparameter C₄, and green fluorescence as measurement parameter F_(ml).

[0178] In one method a number of bead subsets, e.g., subsets 1 through10 (identified as sS1-sS10), are prepared, for example, by using a cellsorter to sort a heterogeneous population to collect a homogeneoussubset or alternatively, by preparing the beads using tightly controlledspecifications to ensure production of a homogeneous subset. Each subsetis distinguishable by its characteristic pattern of classificationparameters C₁, C₂, C₃, and C₄. The beads in each subset are then labeledwith a different antigen such as AgA, AgB, etc. so as to create acollection of labeled subsets as follows: sS1-AgA, sS2-AgB, sS3-AgC,sS4-AgD, sS5-AgE, sS6-AgF, sS7-AgG, sS8-AgH, sS9-AgI, and sS10-AgJ.

[0179] Antigens AgA through AgJ may be attached to the beads by any of anumber of conventional procedures such as by chemical or physicalabsorption as described by Colvin et al., “The Covalent Binding ofEnzymes and Immunoglobulins to Hydrophilic Microspheres” inMicrospheres: Medical and Biological Applications, 1-13, CRC, BocaRaton, Fla., 1988; Cantarero et al., “The Adsorptive Characteristics ofProteins for Polystyrene and Their Significance in Solid-PhaseImmunoassays,” Anal. Biochem., 105, 375-382 (1980); and Illum et al.,“Attachment of Monoclonal Antibodies to Microspheres,” Methods inEnzymol., 112, 67-84 (1985).

[0180] After attachment of antigen to the beads' surface, aliquots fromeach subset are mixed to create a pooled or assay beadset, containingknown amounts of beads within each subset. Preferably, the pooled set isprepared with equal volumes of beads from each subset, so that the setcontains about the same number of beads from each subset.

[0181] The assay beadset may then be incubated with a fluid sample ofinterest, such as serum or plasma, to test for the presence ofantibodies in the fluid that are reactive with antigens on the beads.Such incubation will generally be performed under conditions oftemperature, pH, ionic concentrations, and the like that facilitatespecific reaction of antibodies in the fluid sample with antigen on thebead surface. After a period for binding of antibody, the beads in themixture are centrifuged, washed and incubated (again under controlledconditions) for another period of time with a “secondary” antibody suchas, for example, fluorescein labeled goat anti human immunoglobulin. Thesecondary antibody will bind to and fluorescently label antibodies boundto antigen on the beads. Again after washing (or without washing), thebeads are processed by the flow cytometer and the four classificationparameters forward light scatter, side light scatter, red fluorescence,and orange fluorescence are measured and used to identify the subset towhich each bead in the assay beadset belongs. A simultaneous measurementof green fluorescence (measurement parameter) for each bead allows oneto determine whether the bead has antibody bound to it. Because thesubset to which a bead belongs is correlated with the presence of aparticular antigen, e.g., sS1-AgA, one may readily determine thespecificity of the antibody bound to a bead as a function of the subsetto which it belongs.

[0182] Experimental Example

[0183] Three different antigen-antibody pairs were used in a multiplexexperiment demonstrating the ability to detect the presence or absenceof several antibodies in a single sample. Antigens were coupled to latexmicrospheres via carbodiimide coupling, and the corresponding antibodieswere fluorescently labeled with fluorescein isothiocyanate (greenfluorescence—F_(m)). Each antigen was coupled to a unique microsphere.Baseline data for the fluorescent antibodies and antigen-microspherecomplexes used in this experiment are shown in FIG. 13a. Baseline datafor the three bead subsets of FIG. 13a are given in FIG. 13b.

[0184] The absence of fluorescence (C₂ and C₃) immediately discriminatesthe clear beads (subset 50) from beads in the other two subsets. Subsets45 and 50 were further discriminated by side light scatter (C₁) and redfluorescence (C₃). Linear discriminant functions based on theseobservations and created as described in Section 6.2(c); are shown inFIG. 13c. Accepting only clear beads with side light scatter (C₁)within±0.25 standard deviations of the mean, doublets (two beads stucktogether) were eliminated from the analyses. The remaining beads wereclassified by red fluorescence (C₃) at a midpoint of 59.6. A decisiontree based on the discriminant function table (FIG. 13c) is shown inFIG. 14.

[0185] In this experiment, each of four samples (e.g., blood serum fromfour patients) contained all three antigen-microsphere complexes andeither 1 or 2 different fluorescent antibodies in PBS buffer. Afteraddition of the antibodies, the reactions were incubated at roomtemperature for 45 minutes, and then analyzed on the “FACSCAN” usingside light scatter (C₁), orange fluorescence (C₂), and red fluorescence(C₃) as classification parameters. Green fluorescence was used as themeasurement parameter (F_(m)); an increase in green fluorescence by30-fold indicates a specific interaction between an antigen and itscorresponding fluorescinated antibody. In other words, if a subset'smean measured F_(m) value is greater than 30-fold times that subset'sbaseline F_(m) value, then the target analyte is determined to bepresent. These “interpretive” observations are embodied in theinterpretation table shown in FIG. 13d.

[0186] Once the assay database was built, it was tested by running 5,000beads from each bead subset individually through the system. Afterrejecting 23.8% of the beads as doublets, the remaining crimson beads(subset 18) were classified with 99.88% accuracy. Dark red beads (subset45) were classified with 99.96% accuracy with 22.9% rejected asdoublets. Clear beads (subset 50) were classified with 100% accuracywith 9.4% of the beads rejected as doublets.

[0187] The three bead subsets were pooled to form an assay beadset anddivided into 4 sample tubes and processed by the system shown in FIG. 1.The contents of each sample and the mean measured fluorescence (F_(m))for each bead subset are listed in FIG. 13e. The inventive methodcorrectly identified the antibody or antibodies present in each sample.

[0188] An Experimental Refinement

[0189] In an alternative embodiment using a C₄ (e.g., orangefluorescence) labeled reactant as a classification parameter, a variety(for example five) of protein antigens are employed. Bead subsets arefirst generated based on differences in one or more of C₁, C₂, and C₃.Next, a selected antigen labeled with Cy3NHS (an orange fluorophore) isbound to the beads in each subset. To minimize the measured orangefluorescence coefficient of variation for each bead subset, the beadsare sorted with a high speed cell sorter so that only a narrow range ofantigen (orange fluorophore) is found on each bead within a subset. Careshould be taken to select or prepare the beadset so that different C₄values are measured/obtained for each of the (e.g., five) differentantigens used. In other words, the measured intensity of C₄ for AgAshould differ from the measured intensity of C₄ from AgB, etc. To ensurethat uniformity is achieved, saturation binding with fluoresceinatedmonoclonal antibody is tested—each bead ought to have restricted rangesof both orange and green fluorescence. While the construction ofbeadsets by this method is more laborious, the increase in measurementprecision may be useful and will allow the sampling of fewer beads toarrive at a suitable determination of antibody concentration.

[0190] The assays previously mentioned measure any antibody withspecificity for antigen upon an appropriately labeled bead. The antigencan be quite simple or rather complex and thus, the inventive methodscan measure a highly restricted antibody or a broad array of antibodies.For example, a hexapeptide just large enough to bind to a monoclonalantibody can be employed as antigen or a large protein with manyepitopes can be used. One of ordinary skill will recognize is that thelevel of antibody eventually found associated with the bead (F_(ml)) isa function of the number of epitopes per bead, the concentration ofepitopes, the amount of antibody and the affinity of the antibody andthe valence of the antibody-antigen interaction.

[0191] Displacement Assays

[0192] Assays for many substances in a clinical laboratory are based onthe interference with specific ligand-ligate or antigen-antibodyinteractions. In these assays, one member of the ligand-ligate pair islabeled with the F_(m) fluorophore and one member is immobilized on thebeads. Soluble, unlabeled material (analyte) which may be ligand orligate, is added to the reaction mixture to competitively inhibitinteraction of the labeled component with the immobilized component. Itis usually not important which member of the pair is labeled and whichis immobilized; however, in certain assays, functional advantages maydictate the orientation of the assay.

[0193] In an exemplary assay of this type, each bead subset is modifiedwith an antigen. The antigen-coated beads are then reacted with an F_(m)labeled antibody specific for the antigen on the bead surface.Subsequent addition of a test fluid containing soluble analyte(inhibitor) will displace the F_(m) labeled antibody from the beads indirect proportion to the concentration of the soluble analyte. Astandard curve of known analyte concentrations is used to provideaccurate quantification of analyte in the test sample.

[0194] One of ordinary skill will recognize that the time necessary toachieve equilibrium may be quite lengthy due to the kinetics andassociation constant of the interaction. To lessen the time required forthe assay, the fluid containing the beadset may be subjected todissociating conditions such as a change in pH, ionic strength ortemperature, after mixture of the beadset with the sample to be tested.Alternatively, the F_(m) labeled component may be added to the beadsetafter addition of the test sample. In either case, it is not necessaryfor equilibrium to be achieved to determine analyte concentration if thekinetics and linearity of the assays have been established.

[0195] Additional Experimental Examples

[0196] The following series of experimental examples illustrates how theabove referenced techniques can be used in practice in effectivediagnostic assays. In one embodiment for example, a competitiveinhibition analysis is used to quantitate levels of selected analytes,here IgG, IgA, and IgM. A second experimental refinement demonstratesthe utility of multiplexed assays in epitope mapping of a monoclonalantibody. In one embodiment, that approach involved the use of antibodydetection technology using a fluoresceinated monoclonal antibody incombinatorial epitope screening (e.g. of peptide libraries) to map aparticular epitope to which a monoclonal antibody of interest bound,together with a displacement (competitive inhibition) aspect todemonstrate the specificity of the assay. Also described is a ToRCHassay for screening of human serum for antibodies to a number ofinfectious agents known to pose special hazards to pregnant women.Allergy screening is exemplified by detection of serum IgE against apanel of grass antigens. Yet an additional experimental example reflectsthe ability of the multiplexed assay in pregnancy testing, e.g. intesting for hormones or other analytes commonly elevated duringpregnancy. Each of these examples is set forth below. Simultaneouscompetitive inhibition assay of human immunoglobuling G, A and M levelsin serum

[0197] This example illustrates the determination of multiple analytelevels in a liquid sample simultaneously using competitive inhibitionanalysis. The use of a competitive inhibition assay to accuratelydetermine analyte levels in liquid solutions is a commonly used formatfor many analyte assays. The uniqueness of this assay is thesimultaneous determination of three distinct serum proteins at the sametime in the same tube from one serum sample.

[0198] Immunoglobulins G, A and M are three distinct serum proteinswhose levels are determined by a number of genetic and environmentalfactors in human serum. As changes to these levels may indicate thepresence of disease, clinicians often request assay determinations ofIgG, A and M using conventional techniques. The most common technique isnephelometry that depends upon the absorption of light by precipitatesformed between these immunoglobulins and antibodies made in animals tothe human immunoglobulins. As these immunoglobulins are present in humanserum at fairly high levels, this type of assay is sufficient.Nephelometry however suffers from a number of limitations including theneed for large quantities of reagents, long reaction times forprecipitation to equilibrate and an inability to perform more than onereaction per tube or sample.

[0199] Three competitive inhibition assays are described, one for humanIgG, one for human IgM and one for human IgA using three DifferentiallyFluorescent Microspheres (DFM). Each assay consists of a DFM coated withthe immunoglobulin of choice and a polyclonal, goat anti-human Iglabeled with a green fluorescent molecule (Bodipy). In the absence ofinhibitor, the Bodipy -antibody causes the immunoglobulin (Ig) coatedmicrosphere to emit green fluorescence (F_(m)). In the presence ofinhibitor (soluble Ig), the green signal is reduced. Each assay isbalanced to reflect a sensitivity range near the physiological level ofthe Ig in question at a 1:500 dilution of human serum. Once balanced,the three assays were combined into a multiple analyte format andassayed simultaneously using flow cytometry.

[0200] Antibody Labeling:

[0201] Goat anti-human IgG, goat anti-human IgA, and goat anti-human IgMantibodies (Cappel Division, Organon Teknika, Durham, N.C.) were labeledwith Bodipy FL-CASE (Molecular Probes, Inc., Eugene, Oreg.) usingmethods described by the manufacturer of the Bodipy succinymidyl ester.The resulting Bodipy labeled antibodies were stored in PBS containing 1mg/mL BSA as stabilizer.

[0202] Antigen Conjugation to Microspheres:

[0203] Four DFM (5.5 μM carboxylate, Bangs Laboratories, Inc. (Carmel,Ind.), dyed by Emerald Diagnostics, Inc. (Eugene, Oreg.)) wereconjugated separately to human IgG, human IgA, human IgM (CappelDivision, Organon Teknika, Durham, N.C.) and BSA with a two-step EDCcoupling method (Pierce Chemicals, Rockford, Ill.) using sulfo-NHS tostabilize the amino-reactive intermediate. 100 μL of each bead type(4.2×10⁷ microspheres) was activated for 20 minutes in a total volume of500 μL containing 500 μg of EDC and Sulfo-NHS in 50 mM sodium phosphatebuffer, pH 7.0. The microspheres were washed twice with 500 μL PBS, pH7.4 using centrifugation at 13,400×g for 30 seconds to harvest themicrospheres. Activated, washed beads were suspended in 250 μL of a 0.05mg/mL solution of protein in PBS, pH 7.4. After 1 hour, the microsphereswere blocked by addition of 250 μL of 1.0 mg/mL BSA, 0.02% Tween, 0.2 Mglycine, in PBS, pH 7.4 and incubated for an additional 30 minutes.Protein coated microspheres were washed twice with 500 μL 0.02% Tween20, 1 mg/mL BSA in PBS, pH 7.4 (PBSTB). and stored in PBSTB atapproximately 3,000,000 microspheres/mL. Microsphere concentrations weredetermined using a hemacytometer.

[0204] Determination of Appropriate Ranges of Quantitation for Each IgAssay:

[0205] The normal range of human Ig levels in serum as reported inClinical Chemistry: Principles and Technics, 2nd Edition, Edited by R.J. Henry, D. C. Cannon and J. W. Winkleman are 569-2210 mg/dL for IgG,51-425 mg/dL for IgA and 18-279 mg/dL for IgM. Each inhibition assay wasdesigned to be sensitive to inhibition across these ranges.

[0206] Single Analyte Assay:

[0207] 10 μL of dilutions of a serum calibrator with known Ig levels(Kamiya Biomedical, Thousand Oaks, Calif.) was first mixed with 10 μL ofIg loaded microspheres containing 7,500 beads. Next, 10 μL of theBodipy-labeled Goat Anti-Ig was added and the mixture incubated atambient temperature for 30 minutes. The mixture was diluted to 300 μL inPBSTB and assayed by flow cytometry. For IgG, the Bodipy-labeled goatanti-hIgG was used at 30 μg/mL. For IgA, the Bodipy-labeled goatanti-hIgA was used at 8 μg/mL. For IgM, the Bodipy-labeled goatanti-hIgM was used at 2.5 μg/mL.

[0208] Cross Reactivity Assay:

[0209] Equivalent amounts of each of the four protein loadedmicrospheres were mixed to produce a bead mixture. 10 μL of the beadmixture (7,500 microspheres) was mixed with 10 μL of diluted serumcalibrators of known Ig level. The assay was initiated by addition of 10μL of one of the Bodipy-labeled antibodies “spiked” with a smallquantity of soluble Ig antigen to alleviate the “hook effect”. Themixtures were incubated for 30 minutes, diluted to 300 μL in PBSTB andassayed by flow cytometry. As before for the single analyte assay, theBodipy-labeled goat anti-hIgG was used at 30 μg/mL. For IgA, theBodipy-labeled goat anti-hIgA was used at 8 μg/mL. For IgM, theBodipy-labeled goat anti-hIgM was used at 2.5 μg/mL. The quantities ofantigen “spikes” were 1.6 μg/mL for IgG, 0.6 μg/mL for IgA and 0.4 μg/mLfor IgM.

[0210] Multiple Analyte Assay:

[0211] Equivalent amounts of each of the four protein loadedmicrospheres were mixed to produce a bead mixture. 10 μL of the beadmixture (7,500 microspheres) was mixed with 10 μL of diluted serumcalibrators of known Ig level as well as three other calibrator sera ofknown Ig level to serve for this purpose as unknowns. The assay wasinitiated by addition of 10 μL of a mixture of the three Bodipy-labeledantibodies “spiked” with a small quantity of the three soluble Igantigen to alleviate the “hook effect”. The mixtures were incubated for30 minutes, diluted to 300 μL in PBSTB and assayed by flow cytometry. Asbefore, the Bodipy-labeled goat anti-hIgG was used at 30 μg/mL. For IgA,the Bodipy-labeled goat anti-hIgA was used at 8 μg/mL. For IgM, theBodipy-labeled goat anti-hIgM was used at 2.5 μg/mL. The quantities ofantigen “spikes” were 1.6 μg/mL for IgG, 0.6 μg/mL for IgA and 0.4 μg/mLfor IgM.

[0212] Results

[0213] IgG Single Analyte Assay:

[0214] Results of the single analyte inhibition analysis for IgG levelis shown in Table 1 and FIG. 15A. This assay was designed to be mostsensitive to inhibition in the anticipated range of IgG in human serumat a 1:500 dilution. In FIG. 15A, the area of the inhibition curvebetween the dotted lines, left and right, cover the range ofsensitivity. In this case, the inhibitor was known amounts of human IgGfrom a serum calibrator diluted into human serum containing no IgG, IgAor IgM. Dilutions of the calibrator were then diluted 1:500 in PBSTB andincluded as inhibitor in the assay. The Bodipy-labeled anti-hIgG wasused at 30 μg/mL in PBSTB. 7,500 microspheres were used in thisexperiment and 250 were counted by flow cytometry. Note that as theamount of soluble IgG increased, the degree of inhibition as monitoredby the MIF of F_(m) increased proportionally until saturation of thesystem was achieved. On the other end of the inhibition curve note thatthe lower levels of soluble inhibitor caused an elevation in the MIF ofF_(m) as compared with the negative control (human serum with no Ig).This “hook effect” is common in immunoassay and can be adjusted up ordown the is inhibition curve by adjusting both the amount of antibodyand antigen in the soluble portion of the assay. The “hook effect” wasmost prominent in the IgG assay due to the higher concentrations of bothantigen and antibody per microsphere. This was necessary as IgG is foundin serum at higher concentrations than IgA or IgM.

[0215] IgA Single Analyte Assay:

[0216] Results of single analyte inhibition analysis for IgG level isshown in Table 1 and FIG. 15B. This assay was designed to be mostsensitive to inhibition in the anticipated range of IgA in human serumat a 1:500 dilution. In FIG. 15B, the area of the inhibition curvebetween the dotted lines, left and right, cover the range ofsensitivity. In this case, the inhibitor was known amounts of human IgAfrom a serum calibrator diluted into human serum containing no IgG, IgAor IgM. Dilutions of the calibrator were then diluted 1:500 in PBSTB andincluded as inhibitor in the assay. The Bodipy-labeled anti-hIgA wasused at 8 μg/mL in PBSTB. 7,500 microspheres were used in thisexperiment and 250 were counted by flow cytometry. Note that as theamount of soluble IgA increased, the degree of inhibition as monitoredby the MIF of F_(m) increased proportionally until saturation of thesystem was achieved. On the other end of the inhibition curve note thatthe lower levels of soluble inhibitor cause a slight elevation in theMIF of F_(m) as compared with the negative control (human serum with noIg). The “hook effect” was much less pronounced for both IgA and IgM dueto their lower concentrations in serum.

[0217] IgM Single Analyte Assay:

[0218] Results of single analyte inhibition analysis for IgM level isshown in Table 1 and FIG. 15C. This assay was designed to be mostsensitive to inhibition in the anticipated range of IgM in human serumat a 1:500 dilution. In FIG. 15C, the area of the inhibition curvebetween the dotted lines, left and right, cover the range ofsensitivity. In this case, the inhibitor was known amounts of human IgMfrom a serum calibrator diluted into human serum containing no IgG, IgAor IgM. Dilutions of the calibrator were then diluted 1:500 in PBSTB tobe included as inhibitor in the assay. The Bodipy-labeled anti-hIgM wasused at 2.5 μg/mL in PBSTB. 7,500 microspheres were used in thisexperiment and 250 were counted by flow cytometry. Note that as theamount of soluble IgM increased, the degree of inhibition as monitoredby the MIF of F_(m) increased proportionally until saturation of thesystem was achieved. On the other end of the inhibition curve note thatthe lower levels of soluble inhibitor cause a slight elevation in theMIF of F_(m) as compared with the negative control (PBS with no addedIgM). The “hook effect” is much less pronounced for both IgA and IgM dueto their lower concentrations in serum.

[0219] Cross Reactivity Analysis:

[0220] To determine the cross-reactivity of the various assaycomponents, a multiple analyte assay was performed using only one of thethree Bodipy-labeled antibodies. Equivalent numbers of the IgG, IgA, IgMand BSA beads were mixed to make a GAM mixed bead set. To 10 μL of thebead set (7,500 microspheres) was added 10 μL of dilutions of thecalibrator containing IgG, IgA and IgM. The multiple analyte assay wasthen performed using only one of the Bodipy-labeled anti-IgG, IgA or IgMpreparations rather than a mixture. Table 2 and FIGS. 16A, 16B, and 16Cshow the results of these assays. Results indicated that Anti-IgG-Bodipyonly reacted with DFM-IgG Bodipy and not the IgA or IgM beads. Nocross-reactivity with IgA or IgM was noted and the assay was validatedfor further multiple analyte analysis. Also added to this analysis wasthe antigen “spike”. By adding a small amount of soluble antigen to theprobe antibody solution the “hook effect” can be minimized. Note in theIgG cross-reactivity experiment that the MIF of F_(m) for negativecontrol is higher than the lowest concentration of inhibitor. By spikingthe experiment with 1.6 μg/mL IgG the hook effect has no effect at thelower end of inhibitor range leading to a more accurate assay over theentire dynamic range.

[0221] GAM Simultaneous Analysis:

[0222] Equivalent numbers of the IgG, IgA, IgM and BSA beads were mixedto make a GAM mixed bead set. To 10 μL of the bead set (7,500microspheres) was added 10 μL of dilutions of the calibrator containingIgG, IgA and IgM. Also included were several additional calibrators thatserved as unknowns for the demonstrative purpose of this assay. Themultiple analyte assay was then initiated by adding 10 μL of a mixtureof the Bodipy-labeled anti-IgG, IgA and IgM plus the soluble Ig“spikes”. After a 30 minute, room temperature incubation the reactionmixture was diluted to 300 μL and 1000 microspheres counted by flowcytometry. Tables 3-5 and FIGS. 17-19 show the results of these assays.For each of the inhibition curves produced, a polynomial trendline wasused as a non-linear regression analysis. The fit of this trendline tothe data was demonstrated by the R² correlation factor (1.0 is a perfectfit). The factors of the polynomial formula were used to predict thequantity of inhibitor in each dilution of calibrator and “unknown”serum. The differences between the predicted inhibitor quantities andactual amounts were also included in Tables 3-5. Results indicate thatthis multiple analyte inhibition assay can determine the level of these3 serum proteins with an error of less than 10 %. Coefficients ofvariation (CV) between the triplicate data points indicated that theassay was highly precise (no CV greater, than 6%). Limits ofquantitation for each assay were 400-3000 mg/dL for IgG, 60-455 mg/dLfor IgA, and 36-272 mg/dL for IgM. FIG. 20 shows the results of thethree assays graphically represented on the same graph as all threeassays were performed at the same time in the same tube.

[0223] A multiple analyte, competitive inhibition assay for human serumIgG, IgA, and IgM levels has been developed. This assay, that allows thesimultaneous assay of these three protein levels in serum diluted 1:500,demonstrated excellent sensitivity, precision and accuracy. TABLE 1Single analyte inhibition assays IgG MIF of IgA MIF of IgM MIF of Tube #mg/dL Fm mg/dL Fm mg/dL Fm 1 0 1445 0 1654 0 1765 2 0.026 1500 0.00401645 0.0024 1794 3 0.11 1460 0.016 1729 0.010 1929 4 0.42 1512 0.0641734 0.038 1921 5 1.7 1426 0.26 1733 0.15 1815 6 6.8 1619 1.02 1747 0.611829 7 27.1 1684 4.1 1746 2.4 1833 8 108 1943 16.4 1788 9.8 1807 9 1631898 24.6 1813 14.7 1792 10 244 1885 36.9 1806 22.0 1723 11 366 162455.3 1703 33.0 1704 12 549 1456 83.0 1391 49.6 1446 13 824 998 125 97174.3 1267 14 1235 722 187 558 112 879 15 1853 473 280 336 167 591 162779 350 420 240 251 360 17 4169 313 630 140 376 269 18 6253 316 945 103564 242 19 9380 196 1418 75 847 136 20 14070 165 2127 54 1270 102

[0224] TABLE 2 Cross-reactivity analysis in multiple analyte assay Bead1- Bead 2- Bead 3- Bead 4- hu IgG MIF hu IgA MIF hu IgM MIF MIF Tubemg/dL HuIgG mg/dL HuIgA mg/dL HuIgM BSA 1) GAM Beads reacted withanti-IgG-Bodipy @ 30 μg/mL + Ag spikes. 1 0 1868 0 4 0 6 5 2 400 170260.5 4 36.1 7 5 3 561 1463 84.7 4 50.6 5 5 4 785 1218 119 3 70.8 4 5 51099 880 166 3 99.2 3 5 6 1538 674 233 3 139 3 5 7 2154 549 326 2 194 35 8 3015 450 456 2 272 2 5 2) GAM Beads reacted with anti-IgA-Bodipy @ 8μg/mL + Ag spikes. 11 0 3 0 1800 0 2 3 12 400 2 60.5 1455 36.1 2 3 13561 8 84.7 1225 50.6 1 3 14 785 3 119 930 70.8 1 3 15 1099 2 166 60599.2 1 3 16 1538 2 233 392 139 1 3 17 2154 2 326 278 194 1 3 18 3015 2456 163 272 1 3 3) GAM Beads reacted with anti-IgM-Bodipy @ 2.5 μg/mL +Ag spikes. 21 0 3 0 6 0 1536 2 22 400 3 60.5 9 36.1 1284 2 23 561 3 84.72 50.6 1135 2 24 785 3 119 2 70.8 1011 2 25 1099 2 166 2 99.2 776 2 261538 2 233 1 139 620 2 27 2154 2 326 2 194 463 2 28 3015 2 456 1 272 3302

[0225] TABLE 3 hIgG MIF Average MIF Calculated % Tube # mg/dL of Fm MIFCV mg/dL Difference Multiple analyte IgG inhibition data 1 1917 2 0 19431926 0.6% na na 3 1918 4 1811 5 400.4 1737 1772 1.7% 399.3 0.3% 6 1767 71408 8 560.6 1529 1471 3.4% 566.9 −1.1% 9 1476 10 1250 11 784.8 11631236 4.4% 775.3 1.2% 12 1295 13 852 14 1099 867 862 0.8% 1102.5 −0.3% 15868 16 661 17 1538 726 691 3.9% 1556.1 −1.2% 18 687 19 575 20 2154 575580 1.1% 2126.3 1.3% 21 589 22 461 23 3015 466 468 1.5% 3025.1 −0.3% 24478 “UNKNOWNS” 25 1691 26 446 1657 1657 1.7% 411.3 7.8% 27 1624 28 74929 1243 737 763 3.8% 1316.8 −5.9% 30 804 31 464 32 3045 486 476 1.9%2947.1 3.2% 33 479

[0226] TABLE 4 hIgA MIF Average MIF Calculated % Tube # mg/dL of Fm MIFCV mg/dL Difference Multiple analyte IgA inhibition data 1 1954 2 0 19411952 0.4% na na 3 1960 4 1661 5 60.5 1664 1665 0.2% 60.5 0.0% 6 1669 71222 8 84.7 1391 1307 5.3% 84.7 0.0% 9 1308 10 1055 11 118.6 974 10515.9% 118.6 0.0% 12 1125 13 615 14 166.1 595 606 1.4% 166.1 0.0% 15 60716 376 17 232.5 426 400 5.1% 232.6 0.0% 18 399 19 283 20 325.5 280 2872.9% 325.4 0.0% 21 299 22 193 23 455.7 198 195 1.2% 455.7 0.0% 24 193“UNKNOWNS” 25 1569 26 65 1483 1504 3.1% 68.2 −4.9% 27 1460 28 455 29 187457 477 6.1% 197.2 −5.5% 30 518 31 187 32 454 199 201 5.9% 445.4 1.9% 33216

[0227] TABLE 5 hIgM MIF Average MIF Calculated % Tube # mg/dL of Fm MIFCV mg/dL Difference Multiple analyte IgM inhibition data 1 1566 2 0 16151605 1.8% na na 3 1635 4 1345 5 36.1 1312 1328 1.0% 35.7 1.2% 6 1328 71133 8 50.6 1182 1155 1.8% 52.9 −4.6% 9 1151 10 1038 11 70.8 994 10353.2% 68.1 3.9% 12 1074 13 728 14 99.2 733 735 0.9% 100.7 −1.5% 15 744 16514 17 138.8 585 546 5.4% 138.3 0.4% 18 539 19 424 20 194.4 414 419 1.0%194.0 0.2% 21 418 22 298 23 272.1 339 315 5.6% 272.4 −0.1% 24 307“UNKNOWNS” 25 1266 26 40 1248 1241 1.9% 42.8 −7.0% 27 1209 28 608 29 113621 635 4.7% 116.2 −2.8% 30 677 31 289 32 268 315 306 3.9% 281.0 −4.9%33 313

[0228] Epitope Mapping of a Monoclonal Antibody using Flow Cytometry.

[0229] This example demonstrates the screening of combinatorialchemistry products for a biologically active molecule. The generation ofrandom chemical products for empirical discovery of biologicallysignificant molecules is a method that holds great promise for progressin numerous disciplines of science including biology, pharmacology andmedicine. One general problem with the technique is the screening oflarge numbers of unique molecules for a specific activity. Screeningmethods are required that provide high throughput levels of screeningwith adequate specificity and sensitivity for detection of thebiological event in question.

[0230] An experiment was designed to demonstrate the screening ofpeptides for the epitope of a monoclonal antibody. A monoclonal antibody(MAB 384) was chosen that was produced using the spleen cells of a mousehyper-immunized with a defined peptide (amino acid 67-74) from the aminoacid sequence of human myelin basic protein (MBP). Using the amino acidsequence of this region of MBP, nine overlapping octapeptides weresynthesized that covered the predicted epitope. To the carboxyl terminalend of each peptide, glycine-lysine-biotin residues were added. NineDifferentially Fluorescent Microspheres (DFM) were each coated withavidin and one unique peptide of the set was linked through theavidin-biotin interaction to one unique member of the bead set. Thisresulted in a set of microspheres that contained nine members eachcarrying a unique peptide either flanking or representing the monoclonalantibody's epitope. The bead carrying the epitope peptide was detectedusing the MAB 384 antibody labeled with a green fluorescent tag in amultiple analyte analysis. The detection was shown to be specific forthe peptide in question by competitive inhibition and was not affectedby high levels of free biotin.

[0231] Antibody Labeling:

[0232] MAB 384 (Chemicon International, Inc., Temecula, Calif.) waslabeled with Bodipy FL-X (Molecular Probes, Inc., Eugene, OR) usingmethods described by the manufacturer of the Bodipy succinymidyl ester.Absorbance at 280 nm and 504 nm revealed that the resultingBodipy-labeled antibody had a Bodipy to protein ratio of 3.31 and wasstored in PBS containing 1 mg/mL BSA as stabilizer.

[0233] Avidin Conjugation to Microspheres:

[0234] Nine distinctly dyed DFM (5.5 μM, Bangs Laboratories, Inc.(Carnel, Ind.), dyed by Emerald Diagnostics, Inc. (Eugene, Oreg.)) wereconjugated separately to Neutravidin (deglycosylated avidin) with atwo-step EDC coupling method (Pierce Chemicals, Rockford, Ill.) usingsulfo-NHS to stabilize the amino-reactive intermediate. 20 μL (8.4million microspheres) of each bead type was activated for 20 minutes ina total volume of 100 μL containing 500 μg of EDC and Sulfo-NHS in 50 mMsodium phosphate buffer, pH 7.0. The microspheres were washed twice with100 μL PBS, pH 7.4 using centrifugation at 13,400×g for 30 seconds toharvest the microspheres. Activated, washed beads were suspended in 50μL of a 0.25 mg/mL solution of Neutravidin in PBS, pH 7.4. After 2hours, the microspheres were blocked by addition of 50 μL of 0.2 Mglycine, 0.02% Tween 20 in PBS, pH 7.4 and incubated for an additional30 minutes. Protein coated microspheres were washed twice with 100 μL0.02% Tween 20, 1 mg/mL BSA in PBS, pH 7.4 (PBSTB) and stored in PBSTBat approximately 3,000,000 microspheres/mL as determined byhemocytometer count.

[0235] Peptide Attachment to Microspheres:

[0236] Each of the nine DFM conjugated to Neutravidin were treatedseparately with one of the nine biotinylated peptides. 10 μL ofbiotinylated pepitides at 100-200 ng/mL was mixed with 10 μL ofmicrospheres and reacted for 5 minutes followed by 2×100 μL washes inPBSTB. The peptide loaded microspheres were suspended in 20 μL of PBSTB.

[0237] Single Analyte Assay:

[0238] 10 μL of each of the peptide loaded microspheres was reacted with10 μL of the Bodipy-labeled MAB 384 at 15.5 μg/mL in PBSTB for 1 hour,diluted to 300 μL in PBSTB and assayed using flow cytometry. Negativecontrols included the microspheres without peptide and with the BodipyMAB 384.

[0239] Multiple Analyte Assay:

[0240] 10 μL of each of the 9 peptide loaded microspheres was mixed toproduce a bead set. 10 μL of the set was reacted with 10 μL of theBodipy-labeled MAB 384 at 15.5 μg/mL in PBSTB for 1 hour, diluted to 300μL in PBSTB and assayed using flow cytometry. Negative controls includedthe microsphere set without peptide and treated with the Bodipy MAB 384.

[0241] Competitive Inhibition with Soluble Peptide:

[0242] 10 μL of each of the 9 peptide loaded microspheres was mixed toproduce a bead set. 10 μL of the Bodipy-labeled MAB 384 at 15.5 μg/mL inPBSTB was reacted with 10 μL of soluble peptide containing the epitopesequence HYGSLPQK (SEQ ID NO. 1) at 10 μg/mL and incubated for 1 hr. Themicrosphere set was then treated with peptide absorbed Bodipy-labeledMAB 384 at 15.5 μg/mL for 1 hour, diluted to 300 μL in PBSTB and assayedusing flow cytometry.

[0243] Examination of the Effects of Free Biotin:

[0244] 10 μL of each of the 9 peptide loaded microspheres was mixed toproduce a bead set. 10 μL of the mixture was reacted with 10 μL of 10μg/mL free biotin and incubated for 1 hr. The microsphere set was thentreated with Bodipy-labeled MAB 384 at 15.5 μg/mL for 1 hour, diluted to300 μL in PBSTB and assayed using flow cytometry.

[0245] Results

[0246] Description of Peptides to be Screened:

[0247] The amino acid sequence upstream and downstream from the epitopeof monoclonal antibody MAB 384 (amino acid 67-74, YGSLPQ, SEQ ID NO. 2)was determined using the published amino acid sequence (Roth, H. J., etal., J. Neurosci. Res., 17, 321-328, 1990). The table below shows theamino acid sequence of the nine overlapping peptides produced for thescreening assay. Note that to the carboxy-terminal end of all peptideswas added a glycine (G)-lysine (K)-biotin. 1 GLCNMYKDGK-biotin 2   MYKDSHHPGK-biotin 3       SHHPARTAGK-biotin 4         ARTAHYGSGK-biotin 5             HYGSLPQKGK-biotin 6               LPQKSHGRGK-biotin 7                   SHGRTQDEGK-biotin 8                     TQDENPVVGK-biotin 9                        NPVVHFFKGK-biotin

[0248] Single vs. Multiple Analyte Analysis:

[0249] Each of the nine DFM coated with Neutravidin was reacted for 5minutes with one of the nine biotinylated peptides diluted to 250 ng/mLin PBS. For single analyte analysis, each separate microsphere wasreacted with Bodipy-labeled MAB 384 at 15.5 μg/mL for 60 minutes and themixture assayed using flow cytometry. The Mean Intensity of Fluorescence(MIF) of the green fluorescence channel (F_(m)) is shown for eachpeptide-bead as the darker set of bars in FIG. 21. The darkest barsrepresents single analyte analysis of each bead in the absence ofpeptide as a negative control.

[0250] For multiple analyte analysis, the nine bead-peptides were mixedand then reacted with Bodipy-MAB 384 at 15.5 μg/mL. After 60 minutes,the mixture was assayed using flow cytometry and results (MIF of F_(m))are also shown in FIG. 21. Both assays minus added peptide are shown asa negative control. Results indicated that peptide #5 contained theepitope for MAB 384. Peptides #4 and #6 although containing 3 of theepitope's amino acids showed little reactivity. The multiple and singleanalyte assays provided identical results. Numerical data is shown inTable 6.

[0251] Competitive Inhibition Using Soluble Epitope Peptide:

[0252] To further demonstrate the specificity of the assay, solublepeptide containing the epitope (#5) was used to inhibit the reactionshown in FIG. 21. A 10 μL aliquot of the Bodipy-labeled MAB 384 wasmixed with an equal volume of the epitope containing peptide (HYGSLPQK)at 10 μg/mL. After 1 hour the mixture was reacted with 10 μL of the beadmixture for 1 hour and assayed by flow cytometry. Results shown in FIG.22 reveal that the reaction was significantly inhibited to a MIF ofF_(m) of 53. Numerical data for the inhibition assay is shown in Table7.

[0253] Effects of Free Biotin:

[0254] The high avidity of the biotin-avidin interaction makes itunlikely that the various peptides could be released or exchanged frommicrosphere to microsphere. To demonstrate that such a release orexchange does not occur under strenuous conditions the followingexperiment was performed. A 10 μL aliquot of free biotin at 10 μg/mL (40μM) was incubated with 10 μL of the bead-peptide mixture for 1 hour andthen the microspheres reacted with the MAB 384 Bodipy at 15.5 μg/mL for1 hour and assayed by flow cytometry. Results shown in FIG. 23 indicatethat the free biotin at 10 μg/mL did not displace significant amounts ofthe biotinylated epitope peptide. Numerical data for the inhibitionassay is shown in Table 8.

[0255] This epitope mapping example demonstrates the useful applicationof the instant invention to the area of combinatorial screening. Thepeptide carrying the epitope for the mouse monoclonal antibody screenedin this example was clearly identified in a set of nine peptides. Theidentification was further shown to be specific by competitiveinhibition with soluble epitope peptide. In addition, the stability ofthe avidin-biotin interaction for use with flow cytometry wasdemonstrated in an excess of free biotin. TABLE 6 MIF of Fm PeptideAssayed Single Assayed Multiple Bead plus GL-Biotin Single no peptideMultiple no peptide 70/50 GLCNMYKD 72 66 28 28 60/70 MYKDSHHP 57 48 3636 40/70 SHHPARTA 47 43 36 34 40/50 ARTAHYGS 57 47 35 27 70/70 HYGSLPQK1381 66 1348 25 40/40 LPQKSHGR 43 44 67 25 40/60 SHGRTQDE 42 54 35 2670/60 TQDENPVV 73 70 32 23 70/40 NPVVHFFK 60 60 29 21

[0256] TABLE 7 Peptide Assayed plus GL-Biotin w/ free Biotin GLCNMYKD 4MYKDSHHP 7 SHHPARTA 12 ARTAHYGS 13 HYGSLPQK 53 LPQKSHGR 15 SHGRTQDE 11TQDENPVV 9 NPVVHFFK 17

[0257] TABLE 8 Peptide MIF MIF Bead plus GL-Biotin Multiple w/ Biotin70/50 GLCNMYKD 13 17 60/70 MYKDSHHP 17 19 40/70 SHHPARTA 20 22 40/50ARTAHYGS 20 26 70/70 HYGSLPQK 915 1023 40/40 LPQKSHGR 32 20 40/60SHGRTQDE 19 23 70/60 TQDENPVV 31 34 70/40 NPVVHFFK 31 36

[0258] Multiple Analyte Simultaneous ToRCH Assay for Seroconversion.

[0259] This example demonstrates the utility of this invention in thescreening of human serum for antibodies to infectious disease agents.Screening of serum for antibodies to certain infectious disease agentsis often the only method available to determine if a patient has been,or is infected with the agent in question. For example, a common methodof diagnosing HIV infection is by detection of HIV specific antibodiesin the serum. This phenomenon known as seroconversion is commonlyemployed for diagnosis of several important pathogenic infections. Oneof the most commonly employed assay panels of this type is the ToRCHpanel. ToRCH assays detect both serum IgG and serum IgM responses toToxoplasma gondii, Rubella virus, Cytomegalovirus, and Herpes SimplexVirus Types 1 and 2. The importance of this assay especially to thepregnant woman has been well documented as any one of these infectiousagents is capable of crossing the placental barrier and entering theimmunologically naive fetus. These infectious agents can cause severedamage to the fetus and must be avoided. Currently, all ToRCH panelassays for antibodies specific to each of these pathogens is performedseparately in a unique assay tube or microtiter well. This inventionprovides for a multiple analyte format that allows assay for either IgGor IgM antibodies specific for each of the five pathogens at the sametime in the same tube with the same sample.

[0260] A ToRCH assay using flow cytometry has been developed by couplingpurified antigens of T. gondii, Rubella, CMV and HSV Type 1 and Type 2to five Differentially Fluorescent Microspheres (DFM). The specificityof the assay has been demonstrated by treating this bead set with humanserum calibrators certified to be either positive or negative for allfive agents. After this treatment, the bead set was treated with eitherGoat anti-human IgG-Bodipy or Goat anti-human IgM-Bodipy used to developthe assay. In addition, a third calibrator with known levels ofreactivity to each agent was assayed and the results reported.

[0261] Antibody Labeling:

[0262] Goat anti-human IgG and goat anti-human IgM (Cappel Division,Organon Teknika, Durham, N.C.) were labeled with Bodipy FL-CASE(Molecular Probes, Inc., Eugene, Oreg.) using methods described by themanufacturer of the Bodipy succinymidyl ester. Bodipy-labeled antibodieswere stored in PBS containing 1 mg/mL BSA as stabilizer.

[0263] Antigen Conjugation to Microspheres:

[0264] Five DFM (5.5 μM carboxylate, Bangs Laboratories, Inc., Carmel,Ind., dyed by Emerald Diagnostics, Inc., Eugene, Oreg.) were conjugatedseparately to the five ToRCH antigens (Viral Antigens, Inc.) with atwo-step EDC coupling method (Pierce Chemicals, Rockford, Ill.) usingsulfo-NHS to stabilize the amino-reactive intermediate. All antigenswere dialyzed into PBS to remove any reactive amino groups such assodium azide or glycine. The T. gondii preparation (Chemicon, Inc.,Temecula, Calif.) was sonicated for 2 minutes in PBS, 10 mM EDTA to lysethe tachyzoites. 20 μL (8.4 million microspheres) of each bead type wasactivated for 20 minutes in a total volume of 100 μL containing 500 μgof EDC and Sulfo-NHS in 50 mM sodium phosphate buffer, pH 7.0.Microspheres were washed twice with 200 μL PBS, pH 7.4 usingcentrifugation at 13,400×g for 30 seconds to harvest the microspheres.Activated and washed beads were suspended in 100 μL of antigen at 0.05to 0.15 mg/mL in PBS, pH 7.4. After 2 hours, the microspheres wereblocked by addition of 100 μL of 0.2 M glycine, 0.02% Tween 20 in PBS,pH 7.4 and incubated for an additional 30 minutes. Antigen coatedmicrospheres were washed twice with 200 μL 0.02% Tween 20, 1 mg/mL BSAin PBS, pH 7.4 (PBSTB). and stored in PBSTB at approximately 3,000,000microspheres/mL as determined by hemacytometer count.

[0265] Rubella Assay:

[0266] Rubella antigen loaded microspheres were used to examine severalparameters of the assay in a single analyte format prior to theperformance of multiple analyte assays. 10 μL (30,000 microspheres) ofRubella antigen coated beads were reacted with 10 μL of a 1:10 dilutionof four different Rubella calibrator sera (Consolidated Technologies,Inc., Oak Brook, Ill.) and the mixture incubated for 1 hour. These serawere defined using a standard assay for the anti-Rubella IgG activity bythe manufacturer of the calibrators. The units were defined asInternational Units/ mL or IU/mL. Beads were washed in PBSTB bycentrifugation at 13,400×g for 30 seconds and suspended in 40 μL of a 10μg/mL solution of Bodipy-labeled anti-human IgG. This mixture wasincubated for 1 hour, diluted to 300 μL in PBSTB and assayed using flowcytometry. Negative controls included the microspheres with no serumtreated with the Bodipy-labeled antibodies. In addition one calibratorserum containing 70 IU/mL of anti-Rubella IgG activity was titrated in asingle analyte assay.

[0267] Multiple Analyte Assay for IgG and IgM Activities:

[0268] Equivalent amounts of each of the 5 antigen loaded microsphereswas mixed to produce a ToRCH bead mixture. 10 μL (30,000 microspheres)of the mixture was reacted with 10 μL of a 1:400 dilution of ToRCHcontrol or calibrator sera and incubated for 1 hour. The positive andnegative ToRCH control sera did not have defined units of activity. TheToRCH calibrator, however, did have defined levels of anti-ToRCH IgGactivities as defined by INX and DiaMedix diagnostic instruments. Thesevalues were provided by the manufacturer for the lot of calibratorpurchased. Beads were washed in PBSTB by centrifugation at 13,400×g for30 seconds and suspended in 20 μL of a 40 μg/mL solution ofBodipy-labeled anti-human IgG or IgM. This mixture was incubated for 1hour, diluted to 300 μL in PBSTB and assayed using flow cytometry.Negative controls included the microspheres with no serum treatment andthe microspheres treated with the ToRCH negative control serum. Bothnegative controls were developed with the Bodipy-labeled antibodies.

[0269] Results

[0270] Rubella Assay:

[0271] Rubella coated DFM were reacted with 4 human serum calibratorscontaining known levels of IgG antibodies specific for Rubella virionsdefined by International units (IU/mL). The beads were washed anddeveloped with goat anti-human IgG-Bodipy. Results are shown in Table 9and FIG. 24. Increasing units of anti-Rubella activity were reflected inthe Mean Intensity of Fluorescence (MIF) of F_(m) (green channel).Luminex Units (LU) were defined as the MIF of F_(m) for each data pointminus the MIF of F_(m) for the negative control (no serum) multiplied by0. 1, and are included in Table 9.

[0272] Rubella Calibrator Titration:

[0273] The human serum calibrator containing 70 IU/mL of anti-RubellaIgG was serially diluted in PBSTB and assayed with the Rubella coatedmicrospheres and Bodipy-labeled anti-human IgG. Results shown in Table10 and FIG. 25 show that, as expected, the IgG antibodies specific forRubella were titrated with dilution.

[0274] Multiple Analyte ToRCH Analysis for Serum IgG and IgM:

[0275] Each of the five distinct DFM coated with ToRCH antigens plus oneDFM coated with human serum albumin (Miles, Inc., West Haven, Conn.)were mixed in equal volumes and 10 μL (30,000 microspheres) of themixture reacted for 1 hour with triplicate, 20 μL aliquots of a 1:400dilution of the ToRCH controls as well as the Low ToRCH calibrator. Thecalibrator from Blackhawk Systems, Inc. contained known levels of eachpathogen specific antibody as measured on other diagnostic machines.After washing, one set of triplicates was developed with Bodipy-labeledanti-human IgG and another set with Bodipy-labeled anti-human IgM.Numerical results are shown in Tables 11 and 12. Results are presentedgraphically in FIGS. 26A and 26B. Included in the figures are standarddeviation bars for the triplicate measurements. For both IgG and IgMmeasurements, the ToRCH negative control serum (A96601, tubes #1-3)produced MIF of F_(m) similar to the negative control with no serum(tubes #10-12). The ToRCH positive control serum (A96602, tubes #4-6)demonstrated significant IgG activity to all five pathogens. Conversely,the positive control serum had only slight IgM based reactivity to thefive pathogens. The known levels of anti-ToRCH IgG reactivities for theToRCH Calibrator (A96500, tubes #7-9) were compared to the Luminex unitsof each IgG activity as determined by the multiple analyte analysis.Luminex units were defined by subtracting the negative control serumaverage MIF of F_(m) from the average MIF of F_(m) for each antigen andmultiplying by 0.1. The levels of the ToRCH calibrator were defined bythe manufacturer as a factor of activity for each antigen above thelimit of detection for that antigen on a specific diagnostic machine.These results are listed in Table 13.

[0276] A demonstrative ToRCH assay has been developed to simultaneouslyassay for serum IgG or IgM specific for the five ToRCH pathogens in asingle tube. Results of the assay indicate that it is specific for eachpathogen and is as sensitive as currently available instrument basedassays. The multiple analyte format provides a uniquely powerfultechnology for rapid and less expensive serum testing for seroconversionto ToRCH pathogens as well as other infectious agents diagnosed in thismanner. TABLE 9 Anti-Rubella calibration curve Calibrator IU/mL MIF ofFm LU/mL 360 1419 133 225 1004 91 70 458 37 40 376 28 0 92 0

[0277] TABLE 10 Anti-Rubella calibrator titration 70 IU/mL CalibratorReciprocal of Dilution MIF of Fm 1 4510 4 2554 8 1597 16 954 32 652 64392 128 209 256 121 512 99 0 59

[0278] TABLE 11 IgG ToRCH assay Calibrator MIF of Fm in Triplicate Tube# (1:400) Toxo. Rubella CMV HSV I HSV II HSA 1 A96601 21 9 12 16 17 22 2A96601 22 8 10 17 15 26 3 A96601 25 9 11 14 17 22 4 A96602 647 1786 9561223 664 78 5 A96602 590 1677 967 1511 719 81 6 A96602 620 1670 922 1348611 72 7 A96500 103 38 50 128 64 27 8 A96500 95 43 48 127 58 43 9 A9650087 41 49 127 56 29 10 No Serum 21 7 15 18 13 22 11 No Serum 23 8 11 1519 19 12 No Serum 21 5 12 12 16 23 Calibrator Average MIF of Fm (1:400)Toxo. Rubella CMV HSV I HSV II HSA A96601 23 9 11 16 16 23 A96602 6191711 948 1361 665 77 A96500 95 41 49 127 59 33 No Serum 22 7 13 15 16 21

[0279] TABLE 12 IgM ToRCH assay Calibrator MIF of Fm in Triplicate Tube# (1:400) Toxo. Rubella CMV HSV I HSV II HSA 1 A96601 40 10 17 17 21 162 A96601 36 9 15 15 20 17 3 A96601 39 9 19 18 23 20 4 A96602 68 109 5380 52 27 5 A96602 69 112 56 84 52 23 6 A96602 77 133 81 91 64 60 7A96500 66 15 27 34 26 20 8 A96500 67 15 23 37 29 22 9 A96500 66 15 28 3131 29 10 No Serum 40 9 18 17 21 21 11 No Serum 36 8 20 17 19 16 12 NoSerum 38 8 14 17 19 18 Calibrator Average MIF of Fm (1:400) Toxo.Rubella CMV HSV I HSV II HSA A96601 38 9 17 17 21 18 A96602 71 118 63 8556 37 A96500 66 15 26 34 29 24 No Serum 38 8 17 17 20 18

[0280] TABLE 13 Comparison of known levels of anti-ToRCH IgG for theToRCH calibrator from Blackhawk BioSystems with Luminex Units T. gondiiRubella CMV HSV 1 HSV 2 Diagnostic Machine INX INX INX Diamedix DiaMedixused Factor above Limit 1.7× 2.7× 1.7× 2.5× 1.1× of Detection Units ofActivity 11.3 IU/mL 26.9 IU/mL 24.5 IU/mL 50 EU/mL 22 EU/mL LuminexUnits/mL 7.2 LU/mL 3.2 LU/mL 3.8 LU/mL 11.1 LU/mL 4.3 LU/mL

[0281] Simultaneous Assay of Dog Sera for Allergic IgE andAllergen-Specific IgG

[0282] This example demonstrates the screening of serum for IgEantibodies specific for allergens. Screening of serum for IgE antibodiesspecific to allergens is a viable option for allergy testing as comparedwith skin sensitivity testing. The instant invention provides for aformat that can assay for either IgG or IgE responses to numerousallergens at the same time in the same tube with the same sample and istherefore a uniquely powerful method of screening.

[0283] An allergy assay has been developed including 16 grass allergensin a multiple analyte, simultaneous format. A panel of 16 grassallergens were attached to 16 Differentially Fluorescent Microspheres(DFM) with one grass allergen being coated onto one unique member of thebead set. The allergen bead set was treated with diluted dog serum for 1hour and treated with a solution of either Goat anti-Dog IgE or goatanti-dog IgG-FITC for an additional hour. For the IgE assay, beads werewashed clear of this antibody and the bead set treated with an affinitypurified rabbit anti-goat IgG-FITC antibody as probe.

[0284] Results demonstrate a uniquely powerful method of serum screeningfor allergies that provides a true multiple analyte format, as well assensitivity and specificity.

[0285] Allergen Conjugation to Microspheres:

[0286] Sixteen DFM (5.5 μM carboxylate) were conjugated separately to 16soluble grass allergens (provided by Dr. Bill Mandy, BioMedicalServices, Austin, Tex.) with a two-step EDC coupling method (PierceChemicals, Rockford, Ill.) using sulfo-NHS to stabilize theamino-reactive intermediate. All grass allergens were diluted 1:100 intoPBS, pH 7.4. 20 μL (8.4 million microspheres) of each bead type wasactivated for 20 minutes in a total volume of 100 μL containing 500 μgof EDC and Sulfo-NHS in 50 mM sodium phosphate buffer, pH 7.0.Microspheres were washed twice with 100 μL PBS, pH 7.4 usingcentrifugation at 13,400×g for 30 seconds to harvest the microspheres.Activated, washed beads were suspended in 50 μL of diluted allergen.After 2 hours, the microspheres were blocked by addition of 50 μL of 0.2M glycine, 0.02% Tween 20 in PBS, pH 7.4 and incubated for an additional30 minutes. Protein coated microspheres were washed twice with 100 μL0.02% Tween 20, 1 mg/mL BSA in PBS, pH 7.4 (PBSTB). and stored in PBSTBat approximately 3,000,000 microspheres/mL as determined byhemacytometer count.

[0287] Multiplexed K-9 Grass Allergen IgE Assay:

[0288] Equivalent amounts of each of the 16 grass allergen loadedmicrospheres was mixed to produce a bead mixture. 20 μL (60,000microspheres) of the mixture was reacted with 60 μL of a 1:3 dilution ofdog serum in PBSTB and the mixture incubated for 1 hour. Beads werewashed in 200 μL PBSTB by centrifugation at 13,400×g for 30 seconds andsuspended in 40 μL of a 50 μg/mL solution of anti-dog IgE (provided byDr. Bill Mandy, BioMedical Services, Austin, Tex.). After incubation for1 hour, beads were washed in 200 μL PBSTB by centrifugation at 13,400×gfor 30 seconds. Beads were then treated with 40 μL of rabbit anti-goatIgG-FITC (Sigma, St. Louis, Mo.) at 20 μg/mL. After one hour the beadmixture was diluted to 300 μL in PBSTB and assayed using flow cytometry.Negative controls included the microspheres with dog serum, without thegoat anti-dog IgE and with the rabbit anti-goat IgG-FITC. A negativecontrol of the bead set with no dog serum was also included. Allergenspecific dog IgE was determined by subtraction of the mean intensity offluorescence (MIF) of the green channel (F_(m)) for the negativecontrols for each grass allergen from the MIF of F_(m) for the tubesincluding the goat anti-dog IgE.

[0289] Simultaneous K-9 Grass Allergen IgG Assay:

[0290] Equivalent amounts of each of the 16 grass allergen loadedmicrospheres was mixed to produce a bead mixture. 20 μL (8.4 millionmicrospheres) of the mixture was reacted with 20 μL of a 1:10 dilutionof dog serum in PBSTB and the mixture incubated for 1 hour. Beads werewashed in 200 μL PBSTB by centrifugation at 13,400×g for 30 seconds andsuspended in 25 μL of a 50 82g/mL solution of goat anti-dog IgG-FITC.After one hour the bead mixture was diluted to 300 μL in PBSTB andassayed using flow cytometry. Negative controls included themicrospheres with no dog serum and with the goat anti-dog IgG-FITC.Allergen specific dog IgG was determined by subtraction of the meanintensity of fluorescence (MIF) of the green channel (F_(m)) for thenegative control for each grass allergen from the MIF of F_(m) for thetubes including dog serum.

[0291] Results

[0292] Multiple Analyte Dog Anti-Grass Allergen IgG Assay:

[0293] Grass allergen coated DFM were reacted with 6 dog sera providedby BioMedical Services, Austin, Tex. that had been characterized byELISA for anti-grass allergen IgE. The IgG response to these grassallergens was not measured by BioMedical Services. The beads were washedand developed with goat anti-dog IgG-FITC. Results are shown in FIG. 27.The MIF of F_(m) in the absence of dog serum was subtracted from the MIFof F_(m) for each bead with each dog serum. Two dogs, A96324 and A96326demonstrated relatively high IgG reactivity to most of the grassallergens. Two dogs, A96325 and A96317 demonstrated relatively mediumIgG reactivity to most of the grass allergens. Two dogs, A96319 andA96323 demonstrated relatively low IgG reactivity to most of the grassallergens.

[0294] Multiple Analyte Dog Anti-Grass Allergen IgE Assay:

[0295] Grass allergen coated DFM were reacted with 6 dog sera providedby BioMedical Services, Austin, Tex. that had been characterized byELISA for anti-grass allergen IgE. The beads were washed and treatedwith goat anti-dog IgE for 1 hour. The assay was developed with rabbitanti-goat IgG-FITC. Results are shown in FIG. 28. The MIF of F_(m) inthe absence of dog serum was subtracted from the MIF of F_(m) for eachbead with each dog serum. Two dogs, A96325 and A96326 demonstratedrelatively low reactivity to most of the grass allergens with theexception of Wheat grass and several others for A96326. These resultsagree with the ELISA results provided by BioMedical Services. A96325 wasnegative for 11 grass allergens (only ones tested) and A96326 wasnegative for the same 11 grass allergens except for a “Borderline”result in ELISA against a mixture of Wheat and Quack grass (due to thenon-multiplexed format of ELISA assays, allergens are often mixed toincrease the throughput levels). The other four dog sera demonstratedmedium to high IgE responses to several of the grass allergens. Althoughagreement between ELISA and flow cytometry assay results was notabsolute, the two assays followed the same trends. Dogs with IgEreactivity to grass allergens were detected by both assays.

[0296] Comparison of Multiple Analyte IgG and IgE Results:

[0297] The IgG and IgE anti-grass allergen response to each of the 16allergens was compared by graphing. FIGS. 29-34 demonstrate that therewas no correlation between IgG and IgE response to grass allergens inthe six dogs. Some dogs were low responders for both IgE and IgG, somewere reactive with both immunoglobulin subclasses, and some demonstratedIgE reactivity in a low background of IgG specific for the grassallergens. Examination of the IgG reactivity in a serum could identifythose sera in which s the IgE reactivity could be masked by the high IgGreactivity.

[0298] A demonstrative assay for serum IgG or IgE activity to 16 grassallergens has been developed that allows simultaneous assay of all 16allergens at the same time in the same tube using the same sample.Results with 6 dog sera suggested that IgE anti-grass allergen activityas determined by ELISA was in general agreement with results providedusing flow cytometry. In addition, the ease of determination for IgGanti-grass allergen activity in the six dogs was demonstrated.

[0299] A Simultaneous Immunometric Assay For Human ChorionicGonadotropin and Alpha-Fetoprotein

[0300] This example illustrates the determination of multiple analytelevels in a liquid sample simultaneously by immunometric orcapture-sandwich assay. The use of capture-sandwich assays to accuratelydetermine analyte levels in liquid solutions is a commonly used formatfor many analyte assays. The technique is especially useful for thoseanalytes present in low quantities as the first step serves to captureand thus concentrate the analyte. The uniqueness of this assay is themultiple analyte format allowing the simultaneous determination of twodistinct serum proteins at the same time in the same tube from the sameserum sample.

[0301] Human chorionic gonadotropin (hCG), a gonadotropic hormonesecreted by the placenta, is the primary hormonal marker utilized forpregnancy testing. hCG is elevated both in urine and serum duringpregnancy. Alpha fetoprotein (AFP) is the fetal cell equivalent to humanserum albumin. AFP is elevated in pregnancy and in certain types ofmalignancies. Many clinical fertility or pregnancy test panels includeimmunometric assays for these two serum proteins. Immunometric orcapture-sandwich assays for hCG and AFP were developed separately andthen combined in a multiple analyte format.

[0302] The hCG assay was developed by examining several antibody pairsfor their ability to capture and quantitate hCG levels in solution.First, a monoclonal antibody was coupled through carbodiimide linkage toa carboxylate substituted Differentially Fluorescent Microsphere (DFM).Next, a polyclonal, affinity purified antibody was Bodipy-labeled andused to probe DFM captured hormone. Once this assay was adjusted toinclude physiological sensitive ranges, the process was repeated forAFP. Cross-reactivity of the two assays was examined to demonstrate thatthe two assays would not interfere. The assays were then performedsimultaneously. Commercially available serum calibrators were used todemonstrate that both assays were sensitive in clinically relevantranges and an unknown was include to demonstrate how the two assays worksimultaneously.

[0303] Antibody Labeling:

[0304] The two affinity purified polyclonal anti-hCG (AB633) andanti-AFP (M20077) antibodies (Chemicon, Inc., Temecula, Calif. and MedixDivision, Genzyme, San Carlos, Calif.) were labeled with Bodipy FL-CASE(Molecular Probes, Inc., Eugene, Oreg.) using methods described by themanufacturer of the Bodipy succinymidyl ester. The resultingBodipy-labeled antibodies were stored in PBS containing 1 mg/mL BSA asstabilizer.

[0305] Antibody Conjugation to Microspheres:

[0306] Monoclonal anti-hCG (MAB602) and anti-AFP (S10473) captureantibodies were conjugated to microspheres with a two-step EDC couplingmethod (Pierce Chemicals, Rockford, Ill.) using sulfo-NHS to stabilizethe amino-reactive intermediate. 20 μL ( 8.4 million microspheres) ofeach DFM was activated for 20 minutes in a total volume of 100 μLcontaining 500 μg of EDC and Sulfo-NHS in 50 mM sodium phosphate buffer,pH 7.0. Microspheres were washed twice with 200 μL PBS, pH 7.4 usingcentrifugation at 13,400×g for 30 seconds to harvest the microspheres.Washed, activated beads were suspended in 50 μL of a 0.05 mg/mL solutionof antibody in PBS, pH 7.4. After 2 hours, microspheres were blocked byaddition of 50 μL of 0.5 mg/mL BSA, 0.02% Tween 20 in PBS, pH 7.4 andincubated for an additional 30 minutes. Protein coated microspheres werewashed twice with 200 μL 0.02% Tween 20, 1 mg/mL BSA in PBS, pH 7.4(PBSTB) and stored in PBSTB at approximately 3,000,000 microspheres/mL.Microsphere concentrations were determined using a hemacytometer.

[0307] Antibody Pairs Analysis of Hormone Capture Assay:

[0308] Capture assay antibody pairs were screened by coupling potentialcapture antibodies to microspheres and assaying them using all potentialcombinations of capture antibody-bead/Bodipy-labeled probe antibody.Assays were performed using 10 μL of capture antibody microspheres(approximately 30,000) plus 20 μL of antigen solution at 10 μg/mL inPBSTB for a 1 hour incubation. Beads were washed in PBSTB bycentrifugation at 13,400×g for 30 seconds and suspended in 20 μL of a 25μg/mL solution of Bodipy-labeled probe antibody. This mixture wasincubated for 1 hour, diluted to 300 μL in PBSTB and assayed using flowcytometry.

[0309] Antigen Titration Assay:

[0310] Once an antibody pair was chosen for use, the pair was analyzedfor sensitivity and limit of detection by titration of antigen. Assayswere performed using 10 μL of capture antibody microspheres plus 20 μLof antigen dilutions in PBSTB for a 1 hour incubation. Beads were washedin 200 μL PBSTB by centrifugation at 13,400×g for 30 seconds andsuspended in 20 μL of a 25 μg/mL solution of Bodipy-labeled probeantibody. This mixture was incubated for 1 hour, diluted to 300 μL inPBSTB and assayed using flow cytometry.

[0311] Cross-Reactivity Analysis:

[0312] To examine the possibility of cross-reactivity, 10 μL of MAB 602anti-hCG capture beads (5,000 microspheres) were treated with 20 μLdilutions of hCG or AFP. After 1 hour the beads were washed in 200 μLPBSTB by centrifugation at 13,400×g for 30 seconds and suspended in 20μL of either Bodipy-labeled anti-hCG or Bodipy-labeled anti-AFP at 25μg/mL. Conversely, 10 μL of S-10473 anti-AFP capture beads (5,000microspheres) were treated with 20 μL dilutions of hCG or AFP. After 1hour, beads were washed in 200 μL PBSTB by centrifugation at 13,400×gfor 30 seconds and suspended in 20 μL of either Bodipy-labeled anti-hCGor Bodipy-labeled anti-AFP at 25 μg/mL. Mixtures were incubated for 1hour, diluted to 300 μL in PBSTB and assayed using flow cytometry.

[0313] Washed vs. No-Wash Assay Format:

[0314] An AFP/hCG capture antibody bead mixture was made by mixing equalamounts of the two bead types. In duplicate, 10 μL of this bead mixture(10,000 microspheres) was mixed with 20 μL dilutions of AFP/hCG andincubated for 1 hour. One set of beads were washed in PBSTB bycentrifugation at 13,400×g for 30 seconds and suspended in 20 μL of amixture of Bodipy-labeled anti-hCG and anti-AFP both at 25 μg/mL. Thismixture was incubated for 1 hour, diluted to 300 μL in PBSTB and assayedusing flow cytometry. The second set of beads were treated directly with20 μL of a mixture of Bodipy-labeled anti-hCG and anti-AFP both at 25μg/mL. This mixture representing a homogenous (no-wash) assay was alsoincubated for 1 hour, diluted to 300 μL in PBSTB and assayed using flowcytometry.

[0315] Multiple Analyte Assay:

[0316] Once the AFP and hCG antibody pairs were shown not to cross-reactand were adjusted to provide clinically relevant ranges of sensitivityin a homogenous assay, the assays were performed simultaneously usingcommercially available serum calibrators as the source of AFP and hCGantigens. Equivalent amounts of each of the two capture antibody loadedmicrospheres was mixed to produce an AFP/hCG capture mixture. Intriplicate, 10 μL of this bead mixture (5,000 of each microsphere) wasmixed with 20 μL of three serum calibrators (high, medium and low)containing known levels of AFP and hCG and incubated for 1 hour.Mixtures were treated directly with 20 μL of a blend of Bodipy-labeledanti-hCG and anti-AFP both at 25 μg/mL. Mixtures were incubated for 1hour, diluted to 300 μL in PBSTB and assayed by flow cytometry.

[0317] Results

[0318] Antibody Pair Analysis for hCG Capture Assay:

[0319] For hCG antibody pair analysis, five captureantibody/microspheres were prepared and the identical five antibodieswere Bodipy-labeled to serve as probes. Three of the antibodies werespecific for the alpha sub-unit of hCG and two for the beta sub-unit.The three anti-alpha sub-unit antibody/microspheres were assayed forutility with the two Bodipy-labeled anti-beta hCG antibodies.Conversely, the two anti-beta sub-unit antibody/microspheres wereassayed for utility with the three Bodipy-labeled anti-alpha hCGantibodies. Results of this screen are shown in Table 14 and FIG. 35.The 12 combinations of antibodies are shown with (odd numbers) andwithout (even numbers) hCG at 20 μg/mL. It is apparent that the firsttwo antibody pairs, #1 and #3 demonstrated the highest mean intensity offluorescence (MIF) of the F_(m) (green channel). Further examination ofthese two pairs led to the decision to chose the #3 pair of MAB 602 forcapture antibody and AB633-Bodipy as probe antibody for the hCGcapture/sandwich assay.

[0320] Antigen Titration:

[0321] The MAB 602/AB633 anti-hCG capture system was assayed by hCGtitration to determine if the level of sensitivity required for clinicalassay was achievable. A limit of detection of at least 1 ng hCG/mL wasthe target as this was the level of hCG in the low serum calibrator tobe used later in this project. The results of this antigen titration isshown in Table 15 and FIG. 36. The limit of detection was between 20 and200 pg/mL. This revealed that the MAB602/AB633 anti-hCG antibody pairwas sufficiently sensitive for hCG analysis. Included in this analysiswas MIF of F_(m) measurements from counting of 100 or 1000 microspheres.Results were similar. A similar analysis of antibody pairs and antigentitration for AFP identified an AFP pair that could be furtherdeveloped.

[0322] Cross-Reactivity Assay:

[0323] The MAB 602/AB633 anti-hCG capture system and S-10473/M20077anti-AFP capture system were examined for cross reactivity by assayingeach capture bead with each antigen and Bodipy-labeled antibody. Resultsare shown in Table 16 and FIGS. 37A and 37B. No significantcross-reactivity between the hCG and AFP capture systems was detected.

[0324] No-Wash vs. Washed Assay Format:

[0325] The hCG and AFP assays were performed simultaneously and examinedfor the limit of quantitation or dynamic range in both a washed formatand no wash or homogenous format. Result of these antigen titrations areshown in Table 17 and FIGS. 38A and 38B. Results indicated that thehomogenous format provided sufficient dynamic range for the purposes ofclinical relevance.

[0326] Multiple Analyte hCG/AFP Assay:

[0327] The two assays were performed simultaneously using serumcalibrators of known hCG and AFP levels to generate a standard curve.For each standard curve one serum of unknown hCG and AFP level wasincluded to demonstrate how the assay would determine the level of hCGand AFP in the serum.

[0328] The Randox Tri-level calibrators consisted of three serum sampleswith high, medium and low levels of hCG and AFP documented in mU or U/mLfor hCG and AFP respectively. These calibrators are used in at least 12different diagnostic instruments including those of Abbott (Abbott Park,Ill.), bioMerieux (St. Louis, Mo.), Ciba Corning (Medfield, Mass.),Diagnostics Products (Los Angeles, Calif.), Kodak (Rochester, N.Y.),Syva (San Jose, Calif.), Tosoh (Atlanta, Ga.) and Wallac (Gaithersburg,Md.). Literature with the Randox Tri-Level control listed the units ofeach known analyte as defined by each diagnostic machine. We calculatedthe average of the hCG mU/mL and AFP U/mL for the three calibrators. Inthe case of the hCG, the low and medium calibrators contained 22.8 and26.4 mU/mL which were extremely close considering the distance to thehigh calibrator (436 mU/mL). For this reason, we included a 1:2 dilutionof the high range calibrator into hCG/AFP certified negative serum toproduce a fourth level serum calibrator termed Level 3D. Calibrator 3Dwas only used in construction of the hCG standard curve so each of theassays was effectively defined by three point calibration.

[0329] Table 18 shows the results of this multiple analyte assay. Theassay was performed in triplicate and the average MIF of F_(m) computedfor graphing. Coefficients of variation (CV) for the triplicates wereconsistently less than 10% are shown. Also included in the table are thenumber of microspheres correctly identified by the flow cytometry out ofthe 400 counted per tube. Of the 400 beads counted the expected ratio ofMAB 602 containing 60/40 beads to S-10473 containing 40/60 beads was1:1. Therefore of the 200 beads expected, this was the number of beadscorrectly identified and used to compute the MIF of F_(m) for that datapoint.

[0330]FIGS. 39A and 39B graphically represent the data of Table 18. Forboth hCG and AFP a plot of the MIF vs. the log of antigen concentrationproduced a line that was best fit using a third level polynomialequation. The fit for the hCG curve provided an R² of 1.0 and for AFP anR² of 0.9999 was achieved. Using the polynomial equation, theconcentration of the unknowns was computed. Results of these analysesare seen in Table 18. The unknown serum contained 218.55±6.56 mU/mL ofhCG and 39.59±1.19 U/mL of AFP.

[0331] A demonstrative immunometric assay for hCG and AFP in serum hasbeen developed. Assays were first developed as single analyte or singlebead assays, and optimized with regards to sensitivity, limit ofquantitation and cross-reactivity. The assays were then combined toquantitatively determine multiple analyte levels in a liquid solution inthe same tube from the same sample at the same time. Results, usingcommercially available calibrator sera, has proven that this inventionis effective for this type of quantitative assay. TABLE 14 hCG Conc.Sample Description (μg/mL) MIF of Fm 1 A1-Beads + B1 Ab-BD with hCG 20.08790 2 A1-Beads + B1 Ab-BD with no hCG 0.0 108 3 A2-Beads + B1 Ab-BDwith hCG 20.0 9441 4 A2-Beads + B1 Ab-BD with no hCG 0.0 163 5A3-Beads + B1 Ab-BD with hCG 20.0 3150 6 A3-Beads + B1 Ab-BD with no hCG0.0 2984 7 A1-Beads + B2 Ab-BD with hCG 20.0 2287 8 A1-Beads + B2 Ab-BDwith no hCG 0.0 37 9 A2-Beads + B2 Ab-BD with hCG 20.0 1232 10A2-Beads + B2 Ab-BD with no hCG 0.0 42 11 A3-Beads + B2 Ab-BD with hCG20.0 566 12 A3-Beads + B2 Ab-BD with no hCG 0.0 560 13 B1-Beads + A1Ab-BD with hCG 20.0 70 14 B1-Beads + A1 Ab-BD with no hCG 0.0 23 15B2-Beads + A1 Ab-BD with hCG 20.0 346 16 B2-Beads + A1 Ab-BD with no hCG0.0 20 17 B1-Beads + A2 Ab-BD with hCG 20.0 107 18 B1-Beads + A2 Ab-BDwith no hCG 0.0 33 19 B2-Beads + A2 Ab-BD with hCG 20.0 886 20B2-Beads + A2 Ab-BD with no hCG 0.0 56 21 B1-Beads + A3 Ab-BD with hCG20.0 105 22 B1-Beads + A3 Ab-BD with no hCG 0.0 196 23 B2-Beads + A3Ab-BD with hCG 20.0 143 24 B2-Beads + A3 Ab-BD with no hCG 0.0 609

[0332] TABLE 15 MIF of Fm MIF of Fm Sample hCG Conc. (ng/mL) (1000Beads) (1000 Beads) 1 20000 9337 9222 2 2000 9286 9392 3 200 9233 9400 420 8497 8664 5 2 1286 1382 6 0.2 258 254 7 0.02 120 147 8 0.002 122 1219 0.0002 122 149 10 0 128 111

[0333] TABLE 16A MAB602 BEADS - Anti-hCG Antigen hCG AFP hCG AFP Samp.ng/mL anti-hCG anti-hCG anti-AFP anti-AFP 1 1000.0 792 53 47 52 2 100.0761 47 48 48 3 10.0 530 47 47 48 4 1.0 104 47 48 48 5 0.1 55 52 49 48 60.0 48 71 72 48

[0334] TABLE 16B M20077 BEADS - Anti-AFP Antigen hCG AFP hCG AFP Samp.ng/mL anti-hCG anti-hCG anti-AFP anti-AFP 1 1000.0 99 57 78 348 2 100.054 75 44 356 3 10.0 44 44 45 103 4 1.0 51 50 44 98 5 0.1 42 75 49 44 60.0 43 61 45 45

[0335] TABLE 17 AFP hCG Sample AFP hCG No. ng/mL No Wash Washed ng/mL NoWash Washed 1 1000 379 1481 2000 491 3194 2 500 643 1376 1000 770 3158 3250 956 1205 500 1198 3342 4 125 1063 1052 250 1521 2755 5 62 980 814125 2068 2949 6 31 639 612 62 2417 3200 7 16 359 347 31 2514 3183 8 8190 205 16 2440 2528 9 4 94 108 8 1761 1955 10 2 51 59 4 1122 1300 11 133 35 2 650 547 12 0.5 24 25 1 330 359 13 0.25 17 24 0.5 166 175 14 0 1513 0 15 18

[0336] TABLE 18 hCG capture system AFP capture system Tube hCG AFP MIFof MIF MIF Beads MIF of MIF MIF Beads No. Descript. mU/mL U/mL FL1 AVGCV % IDed FL1 AVG CV % IDed 1 Level 1 23 114 74 98 2 Level 1 22.8 10.727 25.67 7% 130 83 78.00 5% 80 3 Level 1 27 140 77 72 4 Level 2 32 93351 58 5 Level 2 26.4 53.8 34 31.67 6% 85 365 362.67 2% 61 6 Level 2 2994 372 65 7 Level 3D 268 92 535 56 8 Level 3D 218 111.5 276 271.67 1%101 562 552.00 2% 69 9 Level 3D 271 96 559 61 10 Level 3 631 106 1109 4611 Level 3 436 223 601 624.00 3% 99 994 1061.00 5% 38 12 Level 3 640 971080 40 13 Negative 8 99 11 104 14 Negative 10 2 7 7.33 6% 111 13 12.338% 106 15 Negative 7 119 13 95 16 Unknown 270 140 268 67 17 Unknown218.55 39.59 264 272.33 3% 141 274 276.00 3% 81 18 Unknown 283 113 28684

[0337] Multiplexed Beadset Standard Curve Using an Inhibition Assay

[0338] This example provides a demonstration of the measurement ofligand-ligate reactions using a multiplexed beadset standard curve.Commonly for ligand-ligate reactions quantitation, known amounts of theligand or ligate are introduced to the reaction leading to theproduction of a standard curve. Values for unknown samples are comparedto the standard curve and quantified. The true multiple assay capabilityof this invention allows for an additional type of standard to beutilized. A multiplexed beadset standard curve for measuring analyteconcentration is created by using several Differentially FluorescentMicrospheres (DFM) coated with either 1) different amounts of ligand(antigen), or 2) different amounts of ligate (antibody), or 3) differentligates possessing different avidities for the ligand (differentmonoclonal antibodies). We have demonstrated an example of the firsttype of multiple analyte standard curve by developing a competitiveinhibition assay for human IgG.

[0339] Four DFM were coated with human IgG at four differentconcentrations. When probed with goat anti-human IgG-Bodipy the MeanIntensity of Fluorescence (MIF) of F_(m) (green channel) for each beadsubset was different. The MIF of F_(m) correlated with the amount ofhIgG used to coat the beads in each subset. If soluble hIgG was mixedwith the reaction in a competitive manner the MIF of F_(m) was reducedfor each bead as less of the probe antibody was bound to the beads. In anormal standard curve, the signal (MIF of F_(m)) is plotted against theconcentration of the inhibitor. For the multiplexed beadset standardcurve, the slope of the MIF of F_(m) across the beads within a subset isplotted against the concentration of inhibitor. Comparison of the twotypes of standard curves revealed them to be of equivalent value forprediction of an unknown amount of inhibitor.

[0340] Human IgG Conjugation to Microspheres:

[0341] Four DFM (5.5 μM carboxylate, Bangs Laboratories, Inc., Carnel,Ind., dyed by Emerald Diagnostics, Inc., Eugene, Oreg.) were conjugatedseparately to 4 different concentrations of hIgG (Cappel Division,Organon Teknika, Durham, N.C.) with a two-step EDC coupling method(Pierce Chemicals, Rockford, Ill.) using sulfo-NHS to stabilize theamino-reactive intermediate. 20 μL (8.4 million microspheres) of eachbead type was activated for 20 minutes in a total volume of 100 μLcontaining 500 μg of EDC and Sulfo-NHS in 50 mM sodium phosphate buffer,pH 7.0. The microspheres were washed twice with 200 μL PBS, pH 7.4 usingcentrifugation at 13,400×g for 30 seconds to harvest the microspheres.Activated, washed beads were suspended in 50 μL of hIgG at 50, 10, 5,and 1 μg/mL in PBS, pH 7.4. After 2 hours, the microspheres were blockedby addition of 50 μL of 0.2 M glycine, 0.02% Tween 20 in PBS, pH 7.4 andincubated for an additional 30 minutes. Protein coated microspheres werewashed twice with 200 μL 0.02% Tween 20, 1 mg/mL BSA in PBS, pH 7.4(PBSTB). and stored in PBSTB at approximately 3,000,000 microspheres/mLas determined by hemacytometer count.

[0342] Antibody Labeling:

[0343] Goat anti-human IgG (Cappel Division, Organon Teknika, Durham,N.C.) was labeled with Bodipy FL-CASE (Molecular Probes, Inc., Eugene,Oreg.) using methods described by the manufacturer of the Bodipysuccinymidyl ester. The resulting Bodipy-labeled antibody was stored inPBS containing 1 mg/mL BSA as stabilizer.

[0344] Multiplexed Beadset Standard Curve:

[0345] Equivalent amounts of each of the 4 differentially loaded IgGmicrospheres was mixed to produce a bead mixture. 10 μL of the goatanti-hIgG-Bodipy at 25 μg/mL in PBSTB was mixed with 10 μL of a dilutionof hIgG in PBSTB. Immediately 10 μL (30,000 microspheres) of the beadmixture was added and the mixture incubated at room temperature for 30minutes. Beads were diluted to 300 μL in PBSTB and assayed using flowcytometry. A negative control included the microspheres with the goatanti-hIgG-Bodipy with no inhibitor (hIgG). Each bead subset was assignedthe value of a consecutive integer (i.e. the bead subset coupled withthe lowest concentration of IgG was given a value of 1, the next highestconcentration was given a value of 2, etcetera) and those numbersplotted against the MIF of each bead subset at each concentration ofinhibitor tested. The slopes (designated here as inter-bead subsetslopes) were computed using linear regression analysis. The inter-subsetslopes were then plotted against the concentration of inhibitor using alogarithmic scale for the concentration of inhibitor. Results wereplotted as the slope of the MIF of F_(m) across the bead set against thelog of hIgG concentration. Curve fitting was performed using a powerfunction trendline and the R² correlation was reported. For a perfectfit, R²=1.0.

[0346] Common Standard Curve:

[0347] Using the data from the assay described above, a common standardcurve was constructed to compare results with the multiple analytestandard curve. Data from the DFM coated at 50 μg/mL hIgG was utilizedto produce a five-point standard curve by plotting the MIF of F_(m)against the log of hIgG concentration. Curve fitting was performed usinga power function trendline and the R² correlation was reported.

[0348] Results

[0349] Multiplexed Beadset Standard Curve for a Competitive InhibitionAssay:

[0350] Four differentially loaded IgG microspheres were utilized in amultiple beadset competitive inhibition assay for hIgG at five differentconcentrations of soluble inhibitor (hIgG). Results of the assay areshown in Table 19. The inhibition pattern on each bead is plotted inFIG. 40. The inter-bead subset slopes are plotted against the logconcentration of inhibitor in FIG. 41. A Power Trendline in Excel wasused to produce the R² of 0.9933.

[0351] Common Standard Curve Using One Bead of the Multiple AnalyteAssay:

[0352] Data from the 50 μg/mL hIgG bead was utilized to produce afive-point standard curve by plotting the MIF of F_(m) against the logof hIgG concentration. Results are shown in FIG. 42. Curve fitting wasperformed using a Power function trendline and R²=0.9942.

[0353] A novel type of standard curve for ligand-ligate measurement wasdemonstrated. Results suggested that the multiplexed beadset standardcurve was of similar utility as the common multi-point standard curve inquantitation of unknown samples. Advantages of the multiplexed beadsetstandard curve include the inclusion of the standard curve microspheresin each point of a multiplexed beadset assay, and the extension of anassay's dynamic range. This may be achieved by increasing theconcentration range of ligand or ligate on the microspheres or byincreasing the range of avidities for ligand on a series ofmicrospheres. TABLE 19 Inhibitor Bead 1 Bead 2 Bead 3 Bead 4 Samp Conc(μg/mL) 1.0 μg/mL IgG 5 μg/mL IgG 10 μg/mL IgG 50 μg/mL IgG SLOPE 1 10014 77 108 288 85.3 2 50 21 100 162 428 128.3 3 25 40 166 267 844 251.3 412.5 110 463 747 1467 435.5 5 6.25 257 1226 1629 2316 658 6 0 134 7931432 2217 688.8

[0354] Nucleic Acid Measurement

[0355] The power and sensitivity of PCR has prompted its application toa wide variety of analytical problems in which detection of DNA or RNAsequences is required. One major difficulty with the PCR technique isthe cumbersome nature of the methods of measuring the reaction'sproducts-amplified DNA.

[0356] A major advance in this area is here. That advance employs a flowcytometric bead-based hybridization assay which permits the extremelyrapid and accurate detection of genetic sequences of interest. In apreferred embodiment of that invention, a bead to which a nucleic acidsegment of interest has been coupled is provided. A PCR product ofinterest (or any other DNA or cDNA segment) is detected by virtue of itsability to competitively inhibit hybridization between the nucleic acidsegment on the bead and a complementary fluorescent nucleic acid probe.The method is so sensitive and precise as to allow the detection ofsingle point mutations in the PCR product or nucleic acid of interest.Although that method in itself provides a pivotal advance in the art ofanalyzing PCR reaction products, the further discovery of methods ofmultiplexing such an analysis, compounds the method's power andversatility to allow simultaneously analysis of a number of nucleic acidproducts or a number of sequences within a single product in a singlesample.

[0357] The multiplexed DNA analysis method described here can be appliedto detect any PCR product or other DNA of interest for specificpolymorphisms or mutations or for levels of expression, e.g. mRNA. Withthe multiplexed techniques provided by the instant invention,individuals can be screened for the presence of histocompatibilityalleles associated with susceptibility to diseases, mutations associatedwith genetic diseases, autoimmune diseases, or mutations of oncogenesassociated with neoplasia or risk of neoplasia. The analysis of DNAsequences occurs generally as follows:

[0358] 1. A beadset containing subsets of beads coupled to nucleic acidsequences of interest is prepared by coupling a unique synthetic orpurified DNA sequence to the beads within each subset.

[0359] 2. Fluorescent probes complementary to the DNA coupled to eachbead subset are prepared.

[0360] Methods known in the art, e.g., as described in U.S. Pat. No.5,403,711, issued Apr. 4, 1995 and incorporated herein by reference, orother methods may be used to fluorescently label the DNA. Since eachprobe will bind optimally only to its complementary DNA-containingsubset, under the conditions of the assay, the fluorescent probes may beadded to the subsets before or after the subsets are pooled, and beforeor after addition of the DNA test sample(s) of interest.

[0361] 3. Tissue, fluid or other material to be analyzed is obtained,and DNA is purified and/or amplified with PCR as necessary to generatethe DNA products to be tested.

[0362] 4. The DNA samples of interest are then mixed with the pooledbeadset under suitable conditions to allow competitive hybridizationbetween the fluorescent probes and the DNA of interest.

[0363] 5. The beadset is then analyzed by flow cytometry to determinethe reactivity of each bead subset with the DNA sample(s). If the testsample contains a DNA sequence complementary to the DNA of a given beadsubset then that subset will exhibit a decreased F_(m) value relative tothe F_(m) value of beads to which a control DNA has been added. Acomputer executed method in accordance with the current invention candetermine the subset from which each bead is derived, and therefore, theidentity of the DNA sequence on the bead and any change in F_(m).

[0364] Detection of Foreign DNA

[0365] The methods of the present invention find wide utility in thedetection of foreign DNA's in, for example, diagnostic assays. Althoughthe DNA segment to be analyzed can be any DNA sequence, in accordancewith this embodiment the selected segment will be a DNA segment of apathogenic organism such as, but not limited to, bacterial, viral,fungal, mycoplasmal, rickettsial, chlamydial, or protozoal pathogens.The procedure has particular value in detecting infection by pathogensthat are latent in the host, found in small amounts, do not induceinflammatory or immune responses, or are difficult or cumbersome tocultivate in the laboratory. The multiplexed DNA detection method of thepresent invention is likely to find particular utility as a diagnosticassay for analysis of a sample from a patient having clinical symptomsknown to be caused by a variety of organisms using a beadset designed todetect DNAs from the variety of organisms known to cause such symptomsto determine which of such organisms is responsible for the symptoms.DNA would be extracted from tissue, fluid or other sources and analyzedas described above.

[0366] Analysis of Genetic Polymorphisms

[0367] The invention may also be used to measure a variety of geneticpolymorphisms in a target DNA of interest. For example, there areseveral genes in the MHC and many are polymorphic. There are at leasttwo applications in which determination of the alleles at each positionof the MHC is of critical importance. The first is the determination ofhaplotype for transplantation, and the second is determination ofhaplotype as indicator of susceptibility to disease. See Gross et al.,“The Major Histocompatibility Complex-Specific Prolongation of MurineSkin and Cardiac Allograft Survival After In Vivo Depletion of Vβ⁺ TCells,” J. Exp. Med., 177, 35-44 (1993). The MHC complex contains twokinds of polymorphic molecules, Class I genes, HLA A, B and D which have41, 61 and 18 known alleles and Class 10 genes, HLA-DRI,3,4,5 HLA-DQAIand BI HLA-DP, DPA1, DPB1, also with many alleles. Each human can haveup to 6 co-dominant Class I genes and 12 co-dominant Class 10 genes.

[0368] In the case of transplantation, the closer the match between thedonor and recipient the greater the chance of transplant acceptance. Amultiplexed assay in accordance with the invention may be employed toperform tissue typing quickly and accurately to identify suitablematches for transplantation.

[0369] In the situation of disease association, it has been found thatindividuals bearing certain alleles are more prone to some diseases thanthe remainder of the population. The frequency of alleles of the MHCgenes is not equal, and sets of alleles are frequently found (linkagedisequilibrium) so that the identification of the exact set of allelesassociated with many diseases is feasible. As one example,insulin-dependent diabetes mellitus (IDDM) is associated with certainHLA-DQ alleles. The number of alleles of DQ in the population is modestand genetic typing by PCR amplification and hybridization with allelespecific probes has been shown to be practical. See Saiki et al.,“Genetic Analysis of Amplified DNA with Immobilized Sequence-SpecificOligonucleotide Probes,” Proc. Natl. Acad. Sci. U.S.A., 86, 6230- 6234(1989).

[0370] For an assay of MHC in accordance with the invention, DNA isobtained from blood or other extractable source, and amplified withprimers specific for the MHC genes under analysis, for example, HLA-DQA.For a full genotyping of the MHC, several samples of DNA would beamplified with different sets of primers to accommodate the large numberof loci and the high degree of polymorphism. The PCR products are thenscreened for specific alleles using beadsets and fluorescent probes asdescribed above.

[0371] Mutation Analysis of Selected Genes: Screening Procedures

[0372] There are several methodologies for determining and comparing DNAsequences in order to detect mutations which are associated with diseaseor neoplasia. When adapted to a bead-based, multiplexed format inaccordance with the current invention, hybridization analysis allows forthe rapid screening of multiple genetic loci for multiple wild type andmutant sequences.

[0373] In a preferred embodiment of the invention, a given geneticlocus, or multiple loci, can be simultaneously screened for the presenceof wild type or mutant sequences. In the same analysis, multiple knownmutations can be distinguished from each other and from the wild typesequence and uncharacterized mutations. In addition, the homozygosity orheterozygosity of known sequences can be determined.

[0374] A general approach for detecting a DNA mutation in accordancewith this aspect of the invention is as follows. In a first step, asuitable probe for detecting a mutation of interest is selected. In anillustrative embodiment, selected oligonucleotides, representingwild-type and mutant sequences, from a region of a gene known to containa mutation are prepared. Such oligonucleotides are coupled tomicrospheres by techniques known in the art, (e.g., carbodiimidecoupling, or other means) to produce individual aliquots of beads havingknown oligonucleotides coupled thereto. The oligonucleotides must be asufficient length to allow specific hybridization in the assay, e.g.,generally between about 10 and 50 nucleotides, more preferably betweenabout 20 and 30 nucleotides in length. In a preferred embodiment, asaturating amount of the oligonucleotideis bound to the bead.Fluorescent oligonucleotides, complementary to all or part of thesequences attached to each bead, are also prepared.

[0375] Next, PCR primers are selected to amplify that region of the testDNA corresponding to the selected probe, which are then used to amplifythe particular region of DNA in the sample that contains the sequencecorresponding to the oligonucleotide coupled to the beads. Either doublestranded or single stranded PCR techniques may be used. If doublestranded product is produced, the amplified PCR product is made singlestranded by heating to a sufficient temperature to and for a sufficienttime to denature the DNA (e.g., for about 1 to about 5 minutes at about90-95° C. in 2.3×SSC hybridization buffer). The mixture is cooled, andthe beads are added and incubated with the PCR product under conditionssuitable to allow hybridization to occur between the oligonucleotide onthe beads and the PCR product (e.g., at room temperature for about 10minutes). The fluorescent DNA probe may then be added and the entiremixture incubated under hybridization conditions suitable to allowcompetitive hybridization to occur (e.g., 5 minutes at 65° C., thencooling to room temperature over a period of several hours in 2.3×SSCbuffer). As those of skill in the art will recognize, the concentrationsof the PCR product and fluorescent probe to be used may vary and may beadjusted to optimize the reaction.

[0376] In general, the concentrations of PCR product and fluorescentprobe to be used are adjusted so as to optimize the detectable loss offluorescence resulting from competitive inhibition without sacrificingthe ability of the assay to discriminate between perfect complementarityand one or more nucleotide mismatches. In an exemplary assay, theconcentration of PCR product complementary to the oligonucleotide boundto the beads may be on the order of 1 to 10 times the molarconcentration of fluorescent probe used. The fluorescent probe shouldpreferably be added in an amount sufficient to achieve slightly lessthan saturation of the complementary oligonucleotide on the beads inorder to obtain maximum sensitivity for competitive inhibition.

[0377] In a multiplexed assay employing the above principles, beadsetsare separately prepared, pooled, and the bead-based hybridizationanalysis performed. In order to screen a given locus for mutations,beadset subsets are prepared such that subset 1 is coupled to a DNAsegment identical to the wild type sequence, subset 2 is coupled to aDNA segment identical to a known mutation 1 (which may represent asingle or multiple point mutations, deletions or insertions), subset 3is coupled to a DNA segment identical to a second known mutation 2, andso on. The subsets are then mixed to create a pooled beadset.

[0378] When a nucleic acid sample is analyzed with such a beadset, onlythe bead subsets containing sequences identical to the test sample willshow a large decrease in fluorescence (F_(m)). Bead subsets containingunrelated or greatly disparate sequences will show little or no decreasein fluorescence (F_(m)) and bead subsets containing very closely relatedsequences, such as point mutants, will show an intermediate decrease influorescence (F_(m)). Thus, a large decrease in the F_(m) of only subset1 would indicate homozygous wild-type; a large decrease in the F_(m) ofboth subset 1 and subset 2 would indicate heterozygous wild-type/mutant1 and so on. If the test sample is less inhibitory than the perfectlycomplementary sequence for any of the known sequences represented by thesubsets then a new uncharacterized mutation is indicated. The testsample could then be sequenced to characterize the new mutation, andthis sequence information used to construct a new subset for the beadsetto detect the newly discovered mutation.

[0379] The present invention has wide-spread advantages for detection ofany of a number of nucleic acid sequences of interest in the genomic DNAof an individual or organism and has the advantages of being both rapidand extremely accurate in effecting the detection of such mutations. Theinvention will find wide applicability in diagnosis of a number ofgenetically associated disorders as well as in other applications whereidentification of genetic mutations may be important. Exemplary diseasesinclude without limitation, diseases such as cystic fibrosis,generalized myotonia and myotonia congenita, hyperkalemic periodicparalysis, hereditary ovalocytosis, hereditary spherocytosis and glucosemalabsorption; which are associated with mutations in the genes encodingion transporters; multiple endocrine neoplasia, which is associated withmutations in the MEN2a, b, and MEN1 genes; familial medullary thyroidcarcinoma, and Hirschsprung's disease, which are associated withmutations in the ret proto-oncogene; familial hypercholesterolemia,which is associated with mutations in the LDL receptor gene;neurofibromatosis and tuberous sclerosis, which are associated withmutations in the NF1 gene, and NF type 2 gene; breast and ovariancancer, which are associated with mutations in the BRCA1, BRCA2, BRCA3genes; familial adenomatous polyposis, which is associated withmutations in the APC gene; severe combined immunodeficiency, which isassociated with mutations in the adenosine deaminase gene; xerodermapigmentosum, which is associated with mutations in the XPAC gene;Cockayne's syndrome, which is associated with mutations in the ERCC6excision repair gene; fragile X, which is associated with mutations inthe fmrl gene; Duchenne's muscular dystrophy, which is associated withmutations in the Duchenne's muscular dystrophy gene; myotonic dystrophy,which is associated with mutations in the myotonic dystrophy proteinkinase gene; bulbar muscular dystrophy, which is associated withmutations in the androgen receptor genes; Huntington's disease, which isassociated with mutations in the Huntington's gene; Peutz-jegher'ssyndrome; Lesch-Nyhan syndrome, which is associated with mutations inthe HPRT gene; Tay-Sachs disease, which is associated with mutations inthe HEXA gene; congenital adrenal hyperplasia, which is associated withmutations in the steroid 21-hydroxylase gene; primary hypertension,which is associated with mutations in the angiotensin gene; hereditarynon-polyposis, which is associated with mutations in the hNMLH1 gene;colorectal carcinoma, which is associated with mutations in the 2mismatch repair genes; colorectal cancer, which is associated withmutations in the APC gene; forms of Alzheimer's disease which have beenassociated with the apolipoprotein E gene, retinoblastoma, which isassociated with mutations in the Rb gene; Li-Fraumeni syndrome, which isassociated with mutations in the p53 gene; various malignancies anddiseases that are associated with translocations: e.g., in the bcr/abl,bcl-2 gene; chromosomes 11 to 14 and chromosomes 15 to 17transpositions. The references at the end of the specification which areexpressly incorporated herein by reference describe genetic mutationsassociated with certain diseases which may be tested for in accordancewith the invention as well as sequences provided in GENBANK, thecontents of which are also expressly incorporated herein by reference.

[0380] Double Stranded Experiment

[0381] For the purposes of illustration, the two complementary strandsof a double-stranded DNA segment are referred to as strand “A” andstrand “B”. Either strand may be designated “A” or “B”. The wild-type“B” strand oligo (ras codon 12) having the oligonucleotide sequence5′-GCCTACGCCACCAGCTCCAACTAC-3′ (SEQ ID NO. 3) was coupled to 3.0micrometers (μm) latex microspheres (manufactured by InterfacialDynamics, Portland, Oreg.) by carbodiimide coupling. Double strandedcompetitor was prepared by combining equal amounts of both the “A” and“B” strands of either the wild-type or mutant version of the oligo,mutant “B” strand having the sequence 5′-GCCTACGCCACAAGCTCCAACTAC-3′(SEQ ID NO. 4) (ras codon 12) in 5×SSC buffer. Annealing wasaccomplished by heating the mixture to 65° C. for five minutes, thencooling slowly to room temperature. Competitive hybridization wasaccomplished by combining approximately 40 picomoles of thebead-attached oligo (wild-type “B” strand) with the indicated amounts ofdouble stranded competitor in 2.3×SSC buffer at approximately 25° C.Finally, 100 picomoles of the fluorescinated oligo (wild-type “A”strand) was added to the reaction mixture. This mixture was incubatedfor two hours at room temperature, and then diluted with 300 μl ofsaline pH 7.3, and analyzed on the “FACSCAN” (manufactured byBecton-Dickinson Immunocytometry Systems, San Jose, Calif.). The resultsare shown in Table 20 below and in FIGS. 43a through 43 c. TABLE 20Double-Stranded Experimental Results Using Wild-Type “B” OligonucleotidePercent Double Stranded Inhibition (%) Fold Competition Competitor(picomole) Wild-Type Mutant Wild-Type/Mutant 10 20 9 2.2 100 35 12 2.91000 56 17 3.3

[0382] These results clearly show that the DNA containing the singlepoint mutation (“Mutant”) was a detectably less effective inhibitor ofhybridization between the DNA on the beads and the fluorescentoligonucleotide probe at all concentrations of competitor tested.

[0383] Single Stranded Experiment

[0384] The wild-type “B” strand oligo (ras codon 12) was coupled to 3.0μm latex microspheres (manufactured by Interfacial Dynamics) bycarbodiimide coupling. Competitive hybridization was accomplished bycombining approximately 40 picomoles of the bead-attached oligo with 100picomoles of the fluorescinated oligo (wild-type “A” strand) in 2.3×SSCbuffer. Finally, the indicated amounts of single stranded competitor(either mutant or wild-type) were added to two separate aliquots of thereaction mixture. These aliquots were incubated for two hours at roomtemperature, and then diluted with 300 μl of saline pH 7.3. and analyzedon the FACSCAN flow cytometer. The results of these experiments are setforth in Table 21 below and in FIGS. 44a and 44 b. TABLE 21Single-Stranded Experimental Results Percent Single Stranded Inhibition(%) Fold Competition Competitor (picomole) Wild-Type MutantWild-Type/Mutant  100 “A” Strand 14 6 2.4 1000 “A” Strand 25 11 2.3

[0385] These results clearly show that the DNA containing the singlepoint mutation (“Mutant”) was a detectably less effective inhibitor ofhybridization between the DNA on the beads and the florescentoligonucleotide probe at all concentrations of competitor tested.

[0386] Resequencing Analysis of PCR Products Using Multiplexed Analysis.

[0387] This example demonstrates the ability of flow cytometry toperform resequencing analysis of PCR products. As a model system, PCRproducts were derived from the DQA1 gene, in the region of the genewhich determines the major alleles of DQA1. The DQA1 gene represents theDNA coding sequence for the alpha chain of the DQ molecule. DQ isclassified as a class II histocompatibility locus and is expressed inallelic form in all humans. Most individuals are heterozygous for DQA,i.e., they express two different DQA alleles. The determination of DQAalleles is used in identity testing for paternity and forensic purposes.

[0388] Seventeen alleles of DQA1 have been defined by DNA sequencing;however, eight major alleles account for the large majority of thepopulation. These alleles are determined by fourteen unique DNAsequences contained within four regions of the DQA1 gene; all fourregions are contained within a 227 base pair PCR product derived fromhuman genomic DNA.

[0389] Flow cytometry was used to determine the presence or absence ofall fourteen DNA sequences in a PCR product simultaneously in a singlereaction tube, thereby allowing determination of the DQA allelesexpressed in a given sample. The system is based on competitivehybridization between the PCR product and complementary oligonucleotidepairs representing each of the fourteen unique DNA sequences. One strandof each oligonucleotide pair is coupled to a unique subset ofmicrospheres and the complementary strand is labeled with a greenemitting fluorophore. After coupling, the fourteen unique microspheresubsets were pooled to produce the mixed bead set. After addition of thefourteen fluorescent oligonucleotides and the PCR product to thebeadset, the mixture is hybridized and then analyzed by flow cytometry.The ability of the PCR product to inhibit the hybridization of thecomplementary fluorescent oligonucleotides to their respectivemicrosphere subsets is used to determine the DNA sequences, and thus,the allele(s) present in the PCR product.

[0390] Microspheres:

[0391] Carboxylate-modified latex (CML) microspheres of 5.5 micron meandiameter were obtained from Bangs Laboratories, Inc. (Carmel, Ind.). Themicrospheres were differentially dyed with varying concentrations of twofluorescent dyes with orange and red emission spectra to producefourteen unique microsphere subsets.

[0392] Oligonucleotides:

[0393] Fourteen oligonucleotide pairs (complementary strands designated“A” and “B”) corresponding to allelic sequences within the DQA1 gene(Table 22) were synthesized by Oligos, Etc. (Wilsonville, Oreg.). usingstandard automated techniques. Each eighteen-base oligonucleotide wassubstituted at the 5′ end with an amino-terminal linker duringsynthesis.

[0394] Oligonucleotide Coupling to Microspheres:

[0395] The “B” strand of each oligonucleotide pair was coupled to aunique subset of CML microspheres using carbodiimide chemistry. Briefly,0.1 mL of a 1 mM solution of oligonucleotide in 0.1 M MES(2-[N-morpholino]ethanesulfonic acid), pH 4.5 was added to 1.0 mL ofmicrospheres (1% solids) in 0.1 M MES, pH 4.5. To this mixture, 0.05 mLof a 10 mg/mL solution of EDC(1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride) was addedand mixed vigorously. The mixture was incubated for 30 minutes at roomtemperature, followed by another addition of EDC, mixing, and incubationas above. Following the second incubation period, the microspheres werepelleted by centrifugation and resuspended in 0.4 mL of 0.1 M MES, pH4.5 and stored at 4° C.

[0396] Oligonucleotide Labeling:

[0397] The “A” strand of each oligonucleotide pair was fluorescentlylabeled with Bodipy FL-X (6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl) amino)hexanoic acid, succinimidylester) (Molecular Probes, Inc., Eugene, Oreg.). Briefly, a 400 μLsolution containing 20 μM oligonucleotide in 0.1 M sodium bicarbonateand 5% DMSO, pH 8.2 was reacted with 30 μL Bodipy FL-X (10 mg/mL inDMSO) for 16-18 hours at room temperature. The mixture was desalted on aPD 10 column equilibrated in TE (10 mM TrisHCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0) to remove excess unreacted dye andstored at 4° C.

[0398] DNA Extraction:

[0399] Tissue sample (template) DNA was purified using the QIAmp BloodKit (Qiagen, Chatsworth, Calif.) for DNA purification. Briefly, 1×10⁷tissue culture cells or 200 μL whole blood is lysed with Qiagen proteaseand Buffer AL. The lysate is incubated at 70° C. for 10 minutes followedby addition of 210 μL ethanol. The mixture is applied to a QIAmp spincolumn and centrifuged at 8,000×g for 1 minute. The filtrate isdiscarded, 500 μL Buffer AW is added to the column and thecentrifugation is repeated; this step is repeated. The filtrates arediscarded and the DNA is eluted into a new tube by addition of 200 μLBuffer AE, incubation at room temperature for 1 minute, followed bycentrifugation as above.

[0400] Polymerase Chain Reaction (PCR:

[0401] PCR primers designated DQA AMP-A (5′-ATGGTGTAAA CTTGTACCAGT-3′,SEQ ID NO. 5) and DQA AMP-B (5′-TTGGTAGCAG CGGTAGAGTTG-3′, SEQ ID NO. 6)(World Health Organization, 1994) were synthesized by Oligos, Etc.(Wilsonville, Oreg.) using standard automated techniques. PCR wasperformed with reagents (PCR buffer, dNTPs, MgCl₂, and TAQ DNApolymerase) from Life Technologies, Inc.(Gaithersburg, Md.). Thereaction mixture (50 μL) contained 1 μM of each primer, 200 nM dNTPs, 3mM MgCl₂, 4-10 μg/mL DNA template, and 2.5 units TAQ DNA polymerase inPCR buffer. The PCR reaction was performed on an Idaho Technologiesthermal cycler (Idaho Falls, Id.) using and initial step at 94° C. for45 sec, and 32 cycles of 94° C. for 30 sec, 48° C. for 60 sec, and 72°C. for 60 sec followed by a final hold at 72° C. for 7 minutes.Production of the product was verified by agarose electrophoresis andwas quantified by size exclusion chromatography on a Superdex 75 (10/30)column (Pharmacia, Piscataway, N.J.). The PCR product was used withoutpurification.

[0402] Competitive Hybridization Analysis:

[0403] The hybridization reaction was performed in a total volume of 40μL, containing approximately 8,000 of each bead subset for a total ofapproximately 110,000 microspheres, 50 nM of each fluorescentoligonucleotide, and 10-200 nM PCR product, as competitor, inhybridization buffer (3 M trimethyl ammonium chloride, 0.15% sodiumdodecyl sulfate, 3 mM EDTA, and 75 mM TrisHCl, pH 8.0). Briefly, thebeadset mixture, in hybridization buffer, was equilibrated at 55° C. Themixture of fluorescent oligonucleotides and PCR product was denatured ina boiling water bath for 10 minutes followed by quick-chilling on icefor 2 minutes. The microspheres were added, mixed well, and the entirereaction was allowed to hybridize for 30 minutes at 55° C. Followinghybridization, the mixture was diluted to 250 μL using hybridizationbuffer and analyzed by flow cytometry.

[0404] Results

[0405] Microspheres for Multiple Analytes:

[0406]FIG. 45 illustrates the classification, using orange and redfluorescence, of the fourteen microsphere subsets used in the DQA1analysis. Each distinct microsphere subset bears one of the fourteenunique oligonucleotide capture probes on its surface. The level of greenfluorescence associated with each subset, after hybridization with thefluorescent oligonucleotide probes, is also determined simultaneously,and measures the reactivity of the fluorescent oligonucleotides (andtherefore, the reactivity of the PCR product) with each uniqueoligonucleotide sequence.

[0407] Titration of Fluorescent Oligonucleotide:

[0408] To optimize the system for detection of PCR products, fluorescentoligonucleotide was titered in the presence or absence of PCRcompetitor. FIG. 46 illustrates the hybridization of increasingconcentrations of fluorescent oligonucleotide “5503A” to microspherescoupled to oligonucleotide “5503B” in the presence or absence of a 200nM concentration of double-stranded 0301 PCR product which contains the5503 sequence. In the absence of competitor, the level of “5503A” whichhybridizes to the microspheres, detected as FL1, increases in a linearmanner and reaches saturation at approximately 10 nM. In the presence ofcompetitor, the binding curve is shifted to the right indicatinginhibition of “5503A” hybridization.

[0409] Concentration Dependence of Inhibition and Detection of PointMutations:

[0410]FIG. 47 illustrates the inhibition of fluorescent oligonucleotidehybridization by varying concentrations of complementary and pointmutant competitors in the presence of a fixed concentration offluorescent oligonucleotide. The solid lines show the inhibition ofhybridization to bead “3401B” induced by competitors 3401 (u) or 3402(n). The dashed lines show inhibition of hybridization to bead “3402B”induced by competitors 3401(s) or 3402 (l). Even at the lowestcompetitor concentration (10 nM), there is approximately a two-folddifference between the reactivity of the identical sequence versus thepoint mutant.

[0411] Specificity of the Multiple Analyte Assay:

[0412] The specificity of the reaction of each DNA competitor sequencewith the multiplexed microsphere subsets is illustrated in Table 23 andFIG. 48, using double-stranded oligonucleotide competitors. The patternof reactivity is consistent with the homology of the differentoligonucleotides with identical sequences showing maximal reactivity,closely related sequences showing less reactivity, and unrelatedsequences showing little or no reactivity.

[0413] Allele-Specific Reactivity Patterns:

[0414] In order to establish the reactivity patterns of the DQA1 allelesin a model system, simulated alleles were prepared by mixing theoligonucleotides representing the DNA sequences that would be presentwithin a single PCR product for a given allele. FIG. 49 illustrates thetyping of four simulated alleles of DQA1. By comparison to the allelereactivity chart shown in Table 24, it can be seen that each of thesimulated alleles types correctly.

[0415] Typing of Homozygous Genomic DNA:

[0416] To verify the ability of flow cytometry to correctly type PCRproducts prepared from genomic DNA, samples of DNA of known, homozygousDQA1 type were obtained from the UCLA Tissue Typing Laboratory, LosAngeles, Calif. After PCR amplification, these samples were typed usingflow cytometry; the results are shown in FIG. 50. By comparison to theallele reactivity chart (Table 24), it can be seen that the systemcorrectly types these samples.

[0417] Typing of Heterozygous Genomic DNA:

[0418] To determine the ability of multiplexed flow analysis toaccurately type heterozygous DQA1 haplotypes, twenty-five samples ofknown heterozygous DQA1 type were obtained from the UCLA Tissue TypingLaboratory, Los Angeles, Calif. The samples of homozygous DNA used abovewere added to the panel and all of the samples were coded and typed in ablinded study. The data from this study are presented in Table 25. Thelast column of Table 25 entitled “Type” indicates whether the haplotypeindicated by UCLA and the Luminex analysis agreed. In 34 of 35 samples,the haplotypes reported by both laboratories agreed; sample number 19was not typed by the UCLA laboratory, but typed clearly as an 0501/0201heterozygote in the Luminex analysis. Thus, the multiplexed analysis iscapable of typing the DQA1 haplotypes with at least 97% accuracy.

[0419] These studies have demonstrated that flow cytometry can rapidlyand accurately perform resequencing analysis of PCR products. The modelsystem used here required the analysis of fourteen DNA sequences todetermine eight different DQA1 alleles. Flow cytometry was able toperform this analysis in a true simultaneous format, using a singlesample of a single PCR product in a single reaction tube. The entireanalysis, including setup, hybridization, flow analysis, and datacollection and analysis can be accomplished within an hour after PCRamplification of the DNA sample. Thus, it is possible to perform tissuetyping or other genetic analysis in less than three hours afterobtaining a sample of blood, tissue, etcetera, including the timerequired for extraction of DNA and PCR amplification. TABLE 22 DQA1 DNASequences Allele Name Sequence “A” Strand Sequence “B” StrandSpecificities DQA2501 TGGCCAGTACACCCATGA (SEQ ID NO. 7)TCATGGGTGTACTGGCCA (SEQ ID NO. 8) 0101, 9401, 0501 DQA2502TGGCCAGTTCACCCATGA (SEQ ID NO. 9) TCATGGGTGAACTGGCCA (SEQ ID NO. 10)0103, 0201, 0601 DQA2503 TGGGCAGTACAGCCATGA (SEQ ID NO. 11)TCATGGCTGTACTGCCCA (SEQ ID NO. 12) 0301 DQA3401 GAGATGAGGAGTTCTACG (SEQID NO. 13) CGTAGAACTCCTCATCTC (SEQ ID NO. 14) 0101, 0104 DQA3402GAGATGAGCAGTTCTACG (SEQ ID NO. 15) CGTAGAACTGCTCATCTC (SEQ ID NO. 16)0102, 0103, 0501 DQA3403 GAGACGAGCAGTTCTACG (SEQ ID NO. 17)CGTAGAACTGCTCGTCTC (SEQ ID NO. 18) 0401, 0601 DQA3404 GAGACGAGGAGTTCTATG(SEQ ID NO. 19) CATAGAACTCCTCGTCTC (SEQ ID NO. 20) 0201, 0301 DQA4101NACCTGGAGAGGAAGGAGA (SEQ ID NO. 21) TCTCCTTCCTCTCCAGGT (SEQ ID NO. 22)0101, 0102, 0201,0301 DQA4102 ACCTGGAGAAGAAGGAGA (SEQ ID NO. 23)TCTCCTTCTTCTCCAGGT (SEQ ID NO. 24) 0103 DQA4103 ACCTGGGGAGGAAGGAGA (SEQID NO. 25) TCTCCTTCCTCCCCAGGT (SEQ ID NO. 26) 0401, 0501, 0601 DQA5501NTCAGCAAATTTGGAGGTT (SEQ ID NO. 27) AACCTCCAAATTTGCTGA (SEQ ID NO. 28)0101, 0102, 0103 DQA5502N TCCACAGACTTAGATTTG (SEQ ID NO. 29)CAAATCTAAGTCTGTGGA (SEQ ID NO. 30) 0201 DQA5503 TCCGCAGATTTAGAAGAT (SEQID NO. 31) ATCTTCTAAATCTGCGGA (SEQ ID NO. 32) 0301 DQA5504TCAGACAATTTAGATTTG (SEQ ID NO. 33) CAAATCTAAATTGTCTGA (SEQ ID NO. 34)0401, 0501, 0601

[0420] TABLE 23 Specificity of Oligonucleotide Inhibition Bead # Oligo2501 2502 2503 3401 3402 3403 3404 4101 4102 4103 5501 5502 5503 5504none  0%  0%  0%  0%  0%  0%  0%  0%  0%  0%  0%  0%  0%  0% 2501 64%10%  5% −1% −6%  4%  1%  4%  1%  3%  5% −9%  2%  3% 2502 19% 77% −7% −2% 0%  1% −3% −3% −1% −4% −4% −4% −5%  4% 2503  7%  1% 85%  1%  2%  4% −3%−2%  4% −1%  1% −1%  5%  6% 3401 −1% −1% −3% 76%  1%  6%  1%  3%  2%  2% 5% −4%  2%  5% 3402 −1% −8% −12%  14% 83% 11% −5% −5% −3% −10%   0% −7%−4%  1% 3403 −2% −3% −1%  0%  7% 73% −1%  2%  1% −1%  5% −9% −4%  2%3404  5%  5%  4%  6%  2%  8% 62% 10%  9%  6% 10%  0%  7%  9% 4101  6% 4%  6% 10%  5% 10%  6% 79% 18% 22% 31%  5% 12% 12% 4102  0% −1% −4%  0%−3%  5%  0%  8% 79%  3%  7% −1%  5%  4% 4103 −2% 11%  3%  5%  6%  7%  5% 4%  7% 71%  0%  8%  9%  6% 5501 −3%  3%  0%  2%  5%  5%  1% −1%  6%  1%79%  9%  6%  4% 5502  3%  5%  5%  5%  7%  3% −1%  1%  2% −4% −7% 86%  4% 1% 5503 −5%  0% −6%  1%  2%  0% −2% −8%  0% −7% −9% 13% 80% −5% 5504 3%  8%  9%  5%  6%  5%  2%  2%  7%  6%  4% 13%  4% 93%

[0421] TABLE 24 Allele Reactivity Chart Allele Pattern Sequence 0101 (1,1, 1, 1) 2501 3401 4101 5501 0102 (1, 2, 1, 1) 2501 3402 4101 5501 0103(2, 2, 2, 1) 2502 3402 4102 5501 0201 (2, 4, 1, 2) 2502 3404 4101 55020301 (3, 4, 1, 3) 2503 3404 4101 5503 0401 (1, 3, 3, 4) 2501 3403 41035504 0501 (1, 2, 3, 4) 2501 3402 4103 5504 0601 (2, 3, 3, 4) 2502 34034103 5504

[0422] TABLE 25 Blinded typing of Genomic DNA Samples for DQA1 BEADSUBSET 2501 2502 2503 3401 3402 3403 3404 4101 4102 4103 5501 5502 55035504 TYPE 1  4% 25% 35%  7% 69%  8% 58% 36% 61% −1% 78%  2% 75% −8% Y 213% −5% −7% 63% −4% −2% −3% 37%  2% −9% 77%  0%  1% −11%  Y 3 19% −2%40% 66% −3%  0% 65% 51% −2% −2% 78% −2% 76% −14%  Y 4 22% 13% 18% 33%68%  9%  1% −3%  6% 33% −14%   0% −11%  36% Y 5 38%  1% −7% 68% 78%  6%−1% 40% −1% 36% 76% −3% −5% 48% Y 6 −13%  −10%  11% −10%  −10%  −7% 45%21% −4% −12%  −24%  −9% 75% −19%  Y 7 −7%  8% −6% −11%  −8% 19% 27%  7%−5% 16% −27%  31% −6% 30% Y 8 20% −5% −9%  1% 65% 53% −2% 37% −2% 30%76% −2% −7% 43% Y 9 32%  5% −1%  7% 76%  5%  2% 57%  0% −1% 84% −3%−11%  −25%  Y 10 32% 10% 60%  4% 72%  1% 71% 60% −4%  7% 83% −32%  76%−40%  Y 11 27%  7%  5% 10% 70%  5%  2% −14%  −5% 45% −21%  −6% −18%  44%Y 12 29%  3% 57%  8% 71%  6% 67% 60%  0%  3% 84% −4% 77% −29%  Y 13 25% 8%  2% 66% −4% −8% −8% 47% −1% −4% 83% −12%  −22%  −36%  Y 14 16%  8%33% −3%  0% 16% 29% 24%  2% 26% −25%  −7% 47% 17% Y 15  7% 19%  8% −6%26%  0% 31% 26% −5% 28% −31%  36% −21%  23% Y 16 32%  9%  2% 18% 76%  8% 5% −1% −2% 48% −4% −6% −17%  52% Y 17  8%  0% 35%  6% 53%  4% 58% 45% 1% 48% −13%  −1% 77% 41% Y 18  1% −2% 55%  3%  1%  1% 75% 54%  4%  6%−14%  −3% 84% −12%  Y 19 12% 18%  2%  5% 47%  8% 54% 37%  5% 46% −11% 46%  1% 39% ? 20 63% 15%  6% 42% 87% 17% 20% 64% 10% 65% 87%  6%  5% 60%Y 21 44% 62%  8% 23% 76% 13% 73% 70% 11% 20% 88% 64%  4%  1% Y 22 −6%26%  5%  4% −3%  2% 55% 48%  6% −1% −7% 58%  1% −11%  Y 23 56% 67% 13%38% 87% 22% 24% 12% 75% 63% 89% 15% 15% 63% Y 24 15% 25%  5% 60% 58%  7%10% 42% 61% −4% 90% −2% −3% −17%  Y 25 47% 15% 12% 75% 15% 50% 17% 56% 4% 55% 83%  2% −5% 51% Y 26 30%  8% 56% 15% 71%  5% 67% 65%  2%  9% 85%−1% 82% −18%  Y 27 13% 10% 10%  7% 27% 21%  5%  1%  3% 47% −16%   5% −5%51% Y 28 23%  2%  0% 17% 23% 60% 14% −2% −1% 58% −18%  −2% −3% 59% Y 2924%  6% 10% 48% 46%  5% 10% 56%  5%  8% 86%  2% −2% −16%  Y 30 38% 12%11% 73% 14% 48%  7% 55%  6% 55% 84%  1% −7% 50% Y 31 −1% −1% 19%  0% 20%−1% 26% 29% −3% 31% −28%  −4% 70% 31% Y 32 57% 16%  6% 83% 81% 11%  6%59%  7% 60% 86%  9% −1% 53% Y 33 −13%  17% 24%  2% −1% 29% 47% 37% −6%50% −11%   0% 80% 52% Y 34 33%  7%  5% 19% 75%  6% 13% 54%  1%  2% 85%24% −3% −3% Y 35 −11%  19% 31% 10% −14%   0% 70% 46% −4% −2% −57%  44%79% −8% Y

[0423] Measuring Enzymes With Bead-Based Assays

[0424] The invention may also be used in several formats for measurementof enzymes, enzyme inhibitors and other analytes. For example, beadsubsets can be generated that are modified with selected fluorescentsubstrates which can be enzymatically cleaved from the bead, resultingin a loss of fluorescence (F_(m)). Enzymes that can be detected andmeasured using the invention include but are not restricted to,protease, glycosidase, nucleotidase, and oxidoreductase. Any enzyme thatresults in selected bond cleavage can be measured. A cartoon of theaction of enzyme on a bead-bound enzyme is shown in FIG. 51a. An enzymethat acts upon a bead-bound substrate so that the bead-bound substratebecomes fluorescent or loses fluorescence comprises an assay for thelevel of enzyme affecting such a change. FIGS. 51b and 51 c depict thesesituations. Alteration of the substrate could be an oxidation orreduction, alteration of a chemical bond such a hydrolysis or otheralteration of the bead-bound substrate so that the fluorescence of thesubstrate is altered in intensity or spectrum.

[0425] Enzymes that act upon pro-enzymes (convertases) can be measuredusing a bead-bound substrate providing the reaction mixture contains thepro-enzyme and beads bearing a substrate that can be acted upon by theactive form of the enzyme. (Providing the specificity of each activatedenzyme is distinct, a multiplexed assay is achievable in which severalpro-enzymes can be measured at the same time.) The sample is introducedinto a mixture of pro-enzymes under reaction conditions. After a fixedtime interval during which the convertase acts upon the pro-enzyme, thebeadsets specific for each enzyme generated from each pro-enzyme areadded and the newly generated activities measured in a subsequent timeperiod which is terminated when the beadsets are analyzed by flowcytometry. Such a process for a single pro-enzyme to enzyme conversionis illustrated by the cartoon in FIG. 51d.

[0426] The action of the enzyme can be measured in an indirect butfacile manner using a bead bound substrate as depicted in FIG. 51e. Theaction of the enzyme on the bead-bound substrate results in theformation or revelation of a ligate for a fluorescent ligand present inthe reaction mixture. The bead bearing the modified substrate thenbecomes fluorescent by virtue of binding of the fluorescent ligand tothe newly formed ligate. In practice, the enzyme(s) would be added tothe beadset under reactive conditions. After a defined time periodduring which the enzyme acts upon the bead bound substrate, the enzymeaction would be stopped and the fluorescent ligands added and after aperiod for association of ligand with the beadsets, the mixture analyzedby flow cytometry. The fluorescent ligands could be of a singlereactivity or multiple ligands employed, the critical specificity isthat of the enzyme for the substrate.

[0427] The bead-bound substrate may be used to detect the activation ofenzyme when the enzyme requires a cofactor for activity. Under thiscircumstance, the level of the cofactor becomes the limiting componentof the reaction mixture and determination of the level of cofactor canbe measured. Such a configuration is illustrated in FIG. 51f. Thereaction mixture contains the bead-bound substrate as well as theapo-enzyme. After introduction of the analyte (enzyme cofactor), thereaction mixture is held under reactive conditions for a fixed period oftime followed by analysis of the beads by flow cytometry, the level ofcofactor limits the level of enzyme activity. Providing the enzymespresent require different cofactors and have action on differentsubstrate-bearing beadsets, several cofactors could be measured in asingle assay mixture.

[0428] In short, bead-borne substrates can be used as reagent as aresoluble substrates for enzymes. However, because each type of beadbearing a unique substrate can be distinguished, a mixture of beadsubsets can be used to measure several enzyme activities simultaneouslyin the same reaction mixture.

[0429] Fluids that can be analyzed using these techniques includeplasma, serum, tears, mucus, saliva, urine, pleural fluid, spinal fluidand gastric fluid, sweat, semen, vaginal secretions, fluid from ulcersand other surface eruptions, blisters, and abscesses, and extracts oftissues including biopsies of normal, malignant, and suspect tissues. Anassay according to this aspect of the invention proceeds as follows:

[0430] 1. Beads containing reactive surface groups (one of thefollowing: amino, aldehyde, acid chloride, amidine, phenolic hydroxyl,phenyl amine, carboxyl) are obtained that can be discriminated on thebasis of, for example, forward angle light scatter, C₁, right anglelight scatter, C₂, and one of several wavelengths of fluorescence C₃ . .. C_(n) which are designated as orange and red fluorescence, forexample, and comprise a number of subsets.

[0431] 2. Subsets thus obtained are derivatized with a peptide(substrate) having a terminal fluorescent green group, for examplefluorescein (F_(m)).

[0432] 3. Unreacted surface groups and hydrophobic surface of the beadare blocked and the subsets are processed by a particle analyzer andsorter (FACSCAN) and a uniform population of particles are separatedwhich have a low coefficient of variance for F_(m). (e.g., 3%).

[0433] 4. A fluid to be tested is diluted with an appropriate buffer andadded to the beadset mixture to allow enzymes present in the sample toreact with (cleave) their corresponding substrate on the surfaces of thebeads.

[0434] 5. After a defined period of time, the reaction is stopped andthe entire mixture processed by a flow cytometer and results aredetermined.

[0435] The presence of an enzyme in the clinical sample is indicated byloss of fluorescence resulting from the cleavage of the fluorescentF_(m) substrate from the bead surface. Because the beads are analyzed ina very small volume (e.g., about 6 picoliters) as they are passedthrough the flow cytometer's laser beam, interference from freefluorescent molecules (cleaved substrate) will not significantlyinterfere with the assay. This obviates the requirement of washing ofthe beads prior to assay and simplifies the procedure significantly.

[0436] Time

[0437] Time measurement is an important feature of the analysis. Theessence of the measurement of an enzyme activity is a change insubstrate with time. The activity can be determined by setting a periodof time during which the clinical sample is in contact with the beadsusing standard conditions of pH, ionic composition and temperature. Twoalternative processes are available for determination of the bead-boundsubstrate with time, that is the time expired while the enzyme(s) is(are) acting on each beadset(s).

[0438] External Time

[0439] In this configuration, as each bead is measured by the flowcytometer, the time at which each measurement was obtained is recordedalong with the bead's other measurements. Prior to the beginning of theassay, the baseline measurement is determined. Once the enzyme (clinicalsample) is added to the bead mixture, the sample analysis begins. As thebeads proceed through the instrument, the time data collected is used todetermine the length of time that the bead has been exposed to theclinical sample. The F_(m) data collected over the period of the assayis used to determine the rate of change of substrate on the beads(kinetics) and thus the rate readily derived for each bead subset in themixture exposed to the clinical sample.

[0440] Internal Time

[0441] Time can be determined and at the same time a quality controlinternally generated by including a “timer” bead subset that bears asubstrate which is acted on by an enzyme that does not naturally occurin the clinical sample to be tested. The use of non-pathogenic microbialenzymes and substrates with human samples, for example, would suffice.The corresponding “timer” enzyme is added to the dilution buffer so thata known concentration of the “timer” enzyme is present in the buffer.The degree of action of the “timer” enzyme upon the beads in the “timer”subset can be measured as a function of the loss of fluorescence of thebeads in the subset to ensure that proper reaction conditions areachieved. The level of fluorescence of the timer beads can thus be usedas an internal standard and an estimation of time.

[0442] Determination of Enzyme Inhibitors or Regulators

[0443] In addition to direct assay of enzymes, an assay of this type mayalso be used to detect enzyme inhibitors or regulators. In accordancewith this variation, samples being tested for inhibitors are added tothe beadset followed by the corresponding enzymes. If inhibitors arepresent, the measured fluorescent (F_(m)) values will not be decreasedto the same extent as a control containing no inhibitors. In accordancewith FIG. 52, in a similar manner, inhibitors of enzyme activators orbinders of cofactors can be measured.

[0444] The present invention provides numerous advantages and overcomesmany problems associated with prior art techniques of multiplexeddiagnostic and genetic analysis apparatus and methods. It will beappreciated by those of ordinary skill having the benefit of thisdisclosure that numerous variations from the foregoing illustration willbe possible without departing from the inventive concept describedherein. Accordingly, it is the claims set forth below, and not merelythe foregoing illustration, which are intended to define the exclusiverights claimed in this application program.

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1 34 1 8 PRT Homo sapiens 1 His Tyr Gly Ser Leu Pro Gln Lys 1 5 2 6 PRTHomo sapiens 2 Tyr Gly Ser Leu Pro Gln 1 5 3 24 DNA Homo sapiens 3gcctacgcca ccagctccaa ctac 24 4 24 DNA Homo sapiens 4 gcctacgccacaagctccaa ctac 24 5 21 DNA Homo sapiens 5 atggtgtaaa cttgtaccag t 21 621 DNA Homo sapiens 6 ttggtagcag cggtagagtt g 21 7 18 DNA Homo sapiens 7tggccagtac acccatga 18 8 18 DNA Homo sapiens 8 tcatgggtgt actggcca 18 918 DNA Homo sapiens 9 tggccagttc acccatga 18 10 18 DNA Homo sapiens 10tcatgggtga actggcca 18 11 18 DNA Homo sapiens 11 tgggcagtac agccatga 1812 18 DNA Homo sapiens 12 tcatggctgt actgccca 18 13 18 DNA Homo sapiens13 gagatgagga gttctacg 18 14 18 DNA Homo sapiens 14 cgtagaactc ctcatctc18 15 18 DNA Homo sapiens 15 gagatgagca gttctacg 18 16 18 DNA Homosapiens 16 cgtagaactg ctcatctc 18 17 18 DNA Homo sapiens 17 gagacgagcagttctacg 18 18 18 DNA Homo sapiens 18 cgtagaactg ctcgtctc 18 19 18 DNAHomo sapiens 19 gagacgagga gttctatg 18 20 18 DNA Homo sapiens 20catagaactc ctcgtctc 18 21 18 DNA Homo sapiens 21 acctggagag gaaggaga 1822 18 DNA Homo sapiens 22 tctccttcct ctccaggt 18 23 18 DNA Homo sapiens23 acctggagaa gaaggaga 18 24 18 DNA Homo sapiens 24 tctccttctt ctccaggt18 25 18 DNA Homo sapiens 25 acctggggag gaaggaga 18 26 18 DNA Homosapiens 26 tctccttcct ccccaggt 18 27 18 DNA Homo sapiens 27 tcagcaaatttggaggtt 18 28 18 DNA Homo sapiens 28 aacctccaaa tttgctga 18 29 18 DNAHomo sapiens 29 tccacagact tagatttg 18 30 18 DNA Homo sapiens 30caaatctaag tctgtgga 18 31 18 DNA Homo sapiens 31 tccgcagatt tagaagat 1832 18 DNA Homo sapiens 32 atcttctaaa tctgcgga 18 33 18 DNA Homo sapiens33 tcagacaatt tagatttg 18 34 18 DNA Homo sapiens 34 caaatctaaa ttgtctga18

What is claimed is:
 1. A method of preparing a beadset capable ofdetecting a plurality of analytes in a single fluid sample by flowcytometric analysis comprising: (a) obtaining a plurality of subsets ofbeads wherein the beads in each subset are sufficiently homogeneous withrespect to at least three selected classification parameters (C₁, C₂, C₃. . . C_(n)) and sufficiently different in at least one of saidclassification parameters from beads in any other subset so that theprofile of classification parameter values within each subset detectableby flow cytometry is unique; (b) coupling the beads within each subsetto a reactant that will specifically react with a given analyte ofinterest in a fluid to be tested; and (c) mixing the subsets of beads toproduce a beadset, wherein the subset identity and therefore thereactant to which the bead has been coupled is identifiable by flowcytometry based on the unique classification parameter profile of thebeads.
 2. A beadset capable of detecting a plurality of analytes in asingle fluid sample by flow cytometric analysis comprising a pluralityof subsets of beads wherein: (a) the beads in each subset aresufficiently homogeneous with respect to at least three selectedclassification parameters (C₁, C₂, C₃ . . . C_(n)) and sufficientlydifferent in at least one of said classification parameters from beadsin any other subset so that the profile of classification parametervalues within each subset detectable by flow cytometry is unique; (b)wherein the beads within each subset are coupled to a reactant that willspecifically react with a given analyte of interest in a fluid to betested; and (c) wherein said subsets have been mixed to produce thebeadset, characterized in that the subset identity and therefore thereactant to which the bead has been coupled is identifiable based on theunique classification parameter profile of the bead.
 3. A method of flowcytometric analysis capable of detecting a plurality of analytes ofinterest in a single fluid sample comprising: (a) obtaining a beadsetcomprising a plurality of subsets of beads wherein the beads in eachsubset; (1) are sufficiently homogeneous with respect to each of atleast three selected classification parameter (C₁, C₂, C₃ . . . C_(n))values and sufficiently different from beads in any other subset in atleast one of said classification parameter values so that the profile ofclassification parameter values within each subset detectable by flowcytometry is unique; and (2) are coupled to a reactant that willspecifically react with a selected analyte of interest in a fluid to betested; (b) mixing, to produce a reacted bead sample, the beadset withthe fluid to be tested under conditions that will allow reactionsbetween analytes of interest in the fluid and the reactants on the beadsin said set, wherein a reaction between a reactant and an analyte ofinterest on a bead causes a change in the value of a fluorescent signal(F_(m)) emitted from said bead; (c) analyzing the reacted sample by flowcytometry to determine the classification parameter value profile and anF_(m) value of each bead analyzed; (d) identifying the subset to whicheach bead belongs and therefore the reactant on the bead as a functionof the unique profile of classification parameter values; and (e)detecting the presence or absence of a particular analyte of interest insaid sample as a function of the identification in step (d) and a changein the F_(m) values of the beads in each of said subsets in the reactedfluid sample relative to the F_(m) values of the beads in each of saidsubsets not reacted with said fluid.
 4. A method of flow cytometricanalysis capable of detecting a plurality of analytes of interest in asingle fluid sample comprising: (a) obtaining a beadset comprising aplurality of subsets of beads wherein the beads in each subset; (1) aresufficiently homogeneous with respect to each of at least three selectedclassification parameter (C₁, C₂, C₃ . . . C_(n)) values andsufficiently different from beads in any other subset in at least one ofsaid classification parameter values so that the profile ofclassification parameter values within each subset detectable by flowcytometry is unique; and (2) are coupled to a reactant that willspecifically react with a selected analyte of interest in a fluid to betested; (b) mixing, to produce a reacted bead sample, the beadset withthe fluid to be tested under conditions that will allow reactionsbetween analytes of interest in the fluid and the reactants on the beadsin said set; (c) mixing with the reacted bead sample a fluorescent labelunder conditions such that said label will bind to and thereby increasethe value of a fluorescent signal F_(m) emitted from said bead; (d)analyzing the reacted sample containing the fluorescent label by flowcytometry to determine the classification parameter value profile and anF_(m) value of each bead analyzed; (e) identifying the subset to whicheach bead belongs and therefore the reactant on the bead as a functionof the unique profile of classification parameter values; and (f)detecting the presence or absence of a particular analyte of interest insaid sample as a function of the identification in step (e) and anincrease in the F_(m) values of the beads in each of said subsets in thereacted fluid sample relative to the F_(m) values of the beads in eachof said subsets not reacted with said fluid.
 5. A method of flowcytometric analysis capable of detecting a plurality of analytes ofinterest in a single sample comprising: (a) obtaining a beadsetcomprising a plurality of subsets of beads wherein the beads in eachsubset; (1) are sufficiently homogeneous with respect to each of atleast three selected classification parameter (C₁, C₂, C₃ . . . C_(n))values and sufficiently different from beads in any other subset in atleast one of said classification parameter values so that the profile ofclassification parameter values within each subset detectable by flowcytometry is unique, (2) are coupled to a reactant that willspecifically react with a selected analyte of interest in a fluid to betested, and (3) are reacted with a fluorescently labeled compound whichcompetes with said analyte for reaction with said reactant; (b) mixing,to produce a reacted bead sample, the beadset with the fluid to betested under conditions that will allow reactions between analytes ofinterest in the fluid and the reactants on the beads in said set andthereby to allow the analytes to competitively inhibit or displace thefluorescently labeled compounds from said beads, resulting in a decreasein a fluorescent signal F_(m) emitted from a bead with which an analyteof interest in the fluid has reacted; (c) analyzing the reacted sampleby flow cytometry to determine the classification parameter valueprofile and an F_(m) value of each bead analyzed; (d) identifying thesubset to which each bead belongs and therefore the reactant on the beadas a function of the unique profile of classification parameter values;and (e) detecting the presence or absence of a particular analyte ofinterest in said sample as a function of the identification in step (d)and an increase in the F_(m) values of the beads in each of said subsetsin the reacted fluid sample relative to the F_(m) values of the beads ineach of said subsets not reacted with said fluid.
 6. The method of anyone of claims 3, 4, and 5 wherein C₁, C₂, and C₃ are each different andare selected from the group consisting of forward light scatter, sidelight scatter and fluorescence.
 7. The method of any one of claims 3, 4,and 5 wherein n is greater than or equal to 4 and C₁ is forward anglelight scatter, C₂ is side angle light scatter, C₃ is fluorescence at afirst wavelength and C₄ is fluorescence at a second wavelength.
 8. Themethod of claim 7 wherein said first wavelength is red and said secondwavelength is orange.
 9. The method of claim 7 wherein said firstwavelength is red, said second wavelength is orange, and the wavelengthof said F_(m) signal is green.
 10. The method of claim 3 wherein saidanalytes of interest are antigens and said reactants are antibodiesspecifically reactive with said antigens.
 11. The method of claim 3wherein said analytes of interest are antibodies and said reactants areantigens specifically reactive with said antibodies.
 12. The method ofclaim 3 wherein said analytes of interest are antigens selected from thegroup consisting of bacterial, viral, fungal, mycoplasmal, rickettsial,chlamydial and protozoal antigens and said reactants are antibodiesspecifically reactive with said antigens.
 13. The method of claim 3wherein said reactants are antigens selected from the group consistingof bacterial, viral, fungal, mycoplasmal, rickettsial, chlamydial andprotozoal antigens and said analytes of interest are antibodiesspecifically reactive with said antigens.
 14. The method of any one ofclaims 10 or 12 wherein said antigens are antigens borne by pathogenicagents responsible for sexually transmitted disease.
 15. The method ofany of claims 10 or 12 wherein said antigens are antigens borne bypathogenic agents responsible for a pulmonary disorder.
 16. The methodof any of claims 10 or 12 wherein said antigens are antigens borne bypathogenic agents responsible for a gastrointestinal disorder.
 17. Themethod of claim 3 wherein said analytes of interest are substances ofabuse.
 18. The method of claim 3 wherein said analytes of interest aretherapeutic drugs.
 19. The method of claim 3 wherein said analytes ofinterest are antigens or antibodies associated with one or more selectedpathological syndromes.
 20. The method of claim 19 wherein saidsyndromes are selected from the group consisting of malignancy, allergy,autoimmune diseases, and blood borne viruses.
 21. The method of claim 19wherein at least one said syndrome is a cardiovascular disorder.
 22. Themethod of claim 3 wherein said analytes of interest are selected fromthe group consisting of analytes testing for pregnancies and hormones.23. The method of claim 3 wherein said fluorescent signal is emittedfrom fluoresceinated antibodies specific for antibodies coupled to saidbeads in said set.
 24. The method of claim 3 wherein said fluorescentsignal is emitted from a fluoresceinated compound specifically reactivewith an immunoglobulin molecule.
 25. The method of claim 3 wherein saidfluorescent signal is emitted from an agent selected from the groupconsisting of a fluoresceinated anti-immunoglobulin antibody or aspecifically reactive fragment thereof, fluoresceinated protein A, andfluoresceinated protein G.
 26. The method of claim 19 wherein saidanalyte comprises autoantibodies and said antigens comprise oligopeptideepitopes reactive with said autoantibodies, said fluorescent labelscomprise fluorescent monoclonal antibodies reactive with said epitopesand wherein the presence of the analyte autoantibodies is detected as aresult in a decrease of F_(m).
 27. The method of claim 3 wherein saidanalytes are enzymes, said reactants are fluorescently labeledsubstrates for said enzymes, said change in F_(m) results from cleavageof said substrates from said beads.
 28. The method of claim 27 whereinsaid enzymes are selected from the groups consisting of proteases,glycosidases, nucleotidases, oxidoreductases, hydrolyases, esterases,convertases, ligases, transferases, phosphorylases, lyases, lipases,peptidases, dehydrogenases, oxidases, phospholipases, decarboxylases,invertases, aldolases, transaminases, synthetases, and phosphotases. 29.The method of claim 3 wherein the fluid to be tested is selected fromthe group consisting of plasma, serum, tears, mucus, saliva, urine,pleural fluid, spinal fluid and gastric fluid, sweat, semen, vaginalsecretions, fluid from ulcers and other surface eruptions, blisters, andabscesses, and extracts of tissues including biopsies of normal,malignant, and suspect tissues.
 30. A method of flow cytometric analysisfor detection of immunoglobulins in a fluid sample comprising the stepsof: (a) obtaining a beadset comprising a plurality of subsets of beadswherein the beads in each subset; (1) are sufficiently homogeneous withrespect to each of at least three selected classification parameter (C₁,C₂, C₃ . . . C_(n)) values and sufficiently different from beads in anyother subset in at least one of said classification parameter values sothat the profile of classification parameter values within each subsetdetectable by flow cytometry is unique; and (2) are coupled to animmunoglobulin that corresponds to the immunoglobulin to be assayed forin the fluid sample; (b) obtaining a fluorescently labeledimmunoglobulin-binding reagent capable of reacting with theimmunoglobulins to be detected; (c) mixing, to produce a reacted beadsample, the beadset with the fluid sample to be tested and thefluorescently labeled immunoglobulin-binding reagent under conditionsthat will allow competitive binding reactions between theimmunoglobulin-binding reagent and immunoglobulin in the fluid to betested and between the immunoglobulin-binding reagent and theimmunoglobulin on the beads in said set, wherein a reaction between abead-bound immunoglobulin and the fluorescently labeledimmunoglobulin-binding reagent causes an increase in the value of afluorescent signal (F_(m)) emitted from said bead; (d) analyzing thereacted sample by flow cytometry to determine the classificationparameter value profile and an F_(m) value of each bead analyzed; (e)identifying the subset to which each bead belongs and therefore theimmunoglobulin on the bead as a function of the unique profile ofclassification parameter values; and (f) detecting a correspondingimmunoglobulin in said sample as a function of the identification instep (e) and a change in the F_(m) values of the beads in each of saidsubsets in the reacted fluid sample relative to the F_(m) values of thebeads in each of said subsets not reacted with said fluid.
 31. Themethod of claim 30 wherein said immunoglobulins to be detected areimmunoglobulins belonging to different immunoglobulin classes.
 32. Themethod of claim 31 wherein said classes are selected from the groupconsisting of IgG, IgM, IgA, and IgE.
 33. The method of claim 32 whereinsaid immunoglobulins to be detected are immunoglobulins belonging todifferent immunoglobulin sub-classes.
 34. The method of claim 33 whereinsaid subclasses are selected from the group consisting of human IgG₁,IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂.
 35. A method of flow cytometricanalysis for detection of immunoglobulin specific for a particularepitope of interest in a sample comprising the steps of: (a) obtaining abeadset comprising a plurality of subsets of beads wherein the beads ineach subset; (1) are sufficiently homogeneous with respect to each of atleast three selected classification parameter (C₁, C₂, C₃ . . . C_(n))values and sufficiently different from beads in any other subset in atleast one of said classification parameter values so that the profile ofclassification parameter values within each subset detectable by flowcytometry is unique; and (2) are coupled to a monoclonal antibodypreparation which is specific for an epitope that is the same epitope asthat epitope which binds to an immunoglobulin to be assayed for; (b)obtaining a plurality of fluorescently labeled reagents wherein each ofsaid reagents bears an epitope to which the monoclonal antibodypreparation coupled to the beads within a subset binds; (c) mixing, toproduce a reacted bead sample, the beadset with the fluid sample to betested and the fluorescently labeled reagents under conditions that willallow competitive binding reactions between the fluorescently labeledreagents and immunoglobulin in the fluid to be tested and between thefluorescently labeled reagents and the monoclonal antibodies on thebeads wherein a reaction between a bead-bound antibody and thefluorescently labeled reagent causes an increase in the value of afluorescent signal (F_(m)) emitted from said bead; (d) analyzing thereacted sample by flow cytometry to determine the classificationparameter value profile and an F_(m) value of each bead analyzed; (e)identifying the subset to which each bead belongs and therefore themonoclonal antibody on the bead as a function of the unique profile ofclassification parameter values; and (f) detecting the presence orabsence of an immunoglobulin in said sample specific for said particularepitope as a function of the identification in step (e) and a change inthe F_(m) values of the beads in each of said subsets in the reactedfluid sample relative to the F_(m) values of the beads in each of saidsubsets not reacted with said fluid.
 36. The method of claim 35 whereinthe epitopes are epitopes located on viral antigens.
 37. The method ofclaim 36 wherein said viral antigen is an antigen from HIV.
 38. A methodof flow cytometric analysis for detection of analytes commonly elevatedin pregnancy in a fluid sample comprising the steps of: (a) obtaining abeadset comprising a plurality of subsets of beads wherein the beads ineach subset; (1) are sufficiently homogeneous with respect to each of atleast three selected classification parameter (C₁, C₂, C₃ . . . C_(n))values and sufficiently different from beads in any other subset in atleast one of said classification parameter values so that the profile ofclassification parameter values within each subset detectable by flowcytometry is unique; and (2) are coupled to an antibody which isspecific for an analyte to be assayed for in the fluid sample; (b)obtaining a plurality of preparations of antibody molecules wherein eachof said preparations contains fluorescently labeled antibodies specificfor an analyte to be assayed for in the fluid sample; (c) mixing, toproduce a reacted bead sample, the beadset with the fluid sample to betested and the fluorescently labeled antibodies under conditions thatwill allow binding reactions between the antibody that is coupled to thebead, the analyte of interest in the fluid to be tested, and thefluorescently labeled antibodies so as to bind said fluorescentantibodies to said beads though binding to said enzymes which are inturn bound to said beads though said bead-bound antibodies and wherein abridging reaction between a bead-bound antibody, the analyte to whichthat antibody binds, and the fluorescently labeled antibody specific forsaid enzyme causes an increase in the value of a fluorescent signal(F_(m)) emitted from said bead; (d) analyzing the reacted sample by flowcytometry to determine the classification parameter value profile and anF_(m) value of each bead analyzed; (e) identifying the subset to whicheach bead belongs and therefore the antibody on the bead as a functionof the unique profile of classification parameter values; and (f)detecting the analyte in said sample as a function of the identificationin step (e) and a change in the F_(m) values of the beads in each ofsaid subsets in the reacted fluid sample relative to values of the beadsin each of said subsets not reacted with said fluid.
 39. The method ofclaim 38 wherein said analytes are selected from the group consisting ofhuman chorionic gonadotropin, alpha fetoprotein, and 3′ estradiol.
 40. Amethod of flow cytometric analysis for determining the epitope to whicha monoclonal antibody binds comprising the steps of: (a) obtaining abeadset comprising a plurality of subsets of beads wherein the beads ineach subset; (1) are sufficiently homogeneous with respect to each of atleast three selected classification parameter (C₁, C₂, C₃ . . . C_(n))values and sufficiently different from beads in any other subset in atleast one of said classification parameter values so that the profile ofclassification parameter values within each subset detectable by flowcytometry is unique; and (2) are coupled to a peptide which provides agiven epitope; (b) obtaining a fluorescently labeled monoclonal antibodyof interest; (c) mixing, to produce a reacted bead sample, the beadsetwith the fluorescently labeled monoclonal antibody under conditions thatwill allow binding reactions between the bead-bound peptide whichprovides the epitope to which the monoclonal antibody is capable ofbinding and said monoclonal antibody, wherein a reaction between abead-bound peptide and the fluorescently labeled monoclonal antibodycauses an increase in the value of a fluorescent signal (F_(m)) emittedfrom said bead; (d) analyzing the reacted sample by flow cytometry todetermine the classification parameter value profile and an F_(m) valueof each bead analyzed; (e) identifying the subset to which each beadbelongs and therefore the peptide on said bead as a function of theunique profile of classification parameter values; and (f) detecting theparticular epitope to which the monoclonal antibody binds as a functionof the identification in step (e) and a change in the F_(m) values ofthe beads in each of said subsets in the sample relative to the F_(m)values of beads not reacted with said monoclonal antibody.
 41. Themethod of claim 40 where said peptides are from 2 -100 amino acids inlength.
 42. A method of flow cytometric assay for antibodies reactivewith given pathogens of interest in a fluid sample comprising the stepsof: (a) obtaining a beadset comprising a plurality of subsets of beadswherein the beads in each subset; (1) are sufficiently homogeneous withrespect to each of at least three selected classification parameter (C₁,C₂, C₃ . . . C_(n)) values and sufficiently different from beads in anyother subset in at least one of said classification parameter values sothat the profile of classification parameter values within each subsetdetectable by flow cytometry is unique; and (2) are coupled to anantigen derived from one of said pathogens of interest: (b) obtaining afluorescently labeled immunoglobulin-reactive reagent; (c) mixing, toproduce a reacted bead sample, the beadset with the fluid sample and thefluorescently labeled immunoglobulin-reactive reagent under conditionsthat will allow binding reactions between the bead-bound antigen andantibody in said sample and the fluorescently labeledimmunoglobulin-reactive reagent wherein a reaction between a bead-boundantigen, antibody in said fluid sample and the fluorescently labeledreagent causes an increase in the value of a fluorescent signal (F_(m))emitted from said bead; (d) analyzing the reacted sample by flowcytometry to determine the classification parameter value profile and anF_(m) value of each bead analyzed; (e) identifying the subset to whicheach bead belongs and therefore the peptide on said bead as a functionof the unique profile of classification parameter values; and (f)detecting the particular epitope to which the monoclonal antibody bindsas a function of the identification in step (e) and a change in theF_(m) values of the beads in each of said subsets in the sample relativeto the F_(m) values of beads not reacted with said fluid sample.
 43. Themethod of claim 42 wherein said antigens comprise one or more of thefollowing antigens: Toxoplasma gondii, Rubella virus, Cytomegalovirus,and Herpes Simplex virus.
 44. The method of claim 43 wherein saidfluorescently labeled immunoglobulin-reactive reagent is anti-Human IgG.45. The method of claim 43 wherein said fluorescently labeledimmunoglobulin-reactive reagent is anti-Human IgM
 46. A method of flowcytometric assay for antibodies reactive with allergens of interest in afluid sample comprising the steps of: (a) obtaining a beadset comprisinga plurality of subsets of beads wherein the beads in each subset; (1)are sufficiently homogeneous with respect to each of at least threeselected classification parameter (C₁, C₂, C₃ . . . C_(n)) values andsufficiently different from beads in any other subset in at least one ofsaid classification parameter values so that the profile ofclassification parameter values within each subset detectable by flowcytometry is unique; and (2) are coupled to an antigen derived from anallergen of interest; (b) obtaining a fluorescently labeled IgE reactivereagent; (c) mixing, to produce a reacted bead sample, the beadset withthe fluid sample and the fluorescently labeled reagent under conditionsthat will allow binding reactions between the bead-bound allergen andantibody in said sample and the fluorescently labeled IgE reactivereagent wherein a reaction between a bead-bound allergen, antibody insaid fluid sample and the fluorescently labeled IgE-reactive reagentcauses an increase in the value of a fluorescent signal (F_(m)) emittedfrom said bead; (d) analyzing the reacted sample by flow cytometry todetermine the classification parameter value profile and an F_(m) valueof each bead analyzed; (e) identifying the subset to which each beadbelongs and therefore the allergen on said bead as a function of theunique profile of classification parameter values; and (f) detecting theparticular epitope to which the monoclonal antibody binds as a functionof the identification in step (e) and a change in the F_(m) values ofthe beads in each of said subsets in the sample relative to the F_(m)values of beads not reacted with said fluid sample.
 47. The method ofclaim 46 wherein said allergen comprise one or more of the followingantigens: Junegrass, Red Top, Brome, Orchard, Timothy, Rye, Fesque,What, Quack, Bermuda, Johnson, Canary, Velvet, Saltgrass, Bahia, andVernal.
 48. The method of claim 46 wherein said fluorescently labeledIgE reactive reagent is anti-human IgE
 49. The method of claim 46wherein said fluorescently labeled IgE reactive reagent is anti-canineIgE.
 50. A method of flow cytometric analysis capable of quantitatingthe concentration of an analyte of interest in a fluid samplecomprising: (a) obtaining a beadset comprising a plurality of subsets ofbeads, wherein the beads in each subset; (1) are sufficientlyhomogeneous with respect to each of at least three selectedclassification parameter (C₁, C₂, C₃, . . . C_(n)) values andsufficiently different from beads in any other subset in at least one ofsaid classification parameter values so that the profile ofclassification parameter values within each subset detectable by flowcytometry is unique; and (2) are coupled to a reactant that willspecifically react with the selected analyte of interest in the sampleto be tested; and wherein the beads in a plurality of said subsets arecoupled to the same reactant but at concentrations which differ amongsaid subsets; (b) mixing, to produce a reacted bead sample, the beadsetwith the fluid sample to be tested under conditions that will allowreactions between the analyte of interest in the fluid sample and thereactants on the beads in said set, wherein a reaction between areactant and an analyte of interest on a bead causes a change in thevalue of a fluorescent signal (F_(m)) emitted from said bead; (c)analyzing the reacted sample by flow cytometry to determine theclassification parameter value profile and an F_(m) value of each beadanalyzed; (d) identifying the subset to which each bead belongs andtherefore the concentration of reactant with which the bead was coupledas a function of the unique profile of classification parameter values;and (e) detecting the concentration of the analyte of interest in saidsample as a function of the identification in step (d) and the F_(m)values of the beads in each of said subsets relative to the F_(m) valuesof a second set of the beads in each of said subsets, wherein said beadsin said second set have not been reacted with said fluid sample but havebeen reacted with a known concentration of the analyte of interest. 51.A method of flow cytometric analysis capable of quantitating theconcentration of an analyte of interest in a fluid sample comprising:(a) obtaining a beadset comprising a plurality of subsets of beadswherein the beads in each subset; (1) are sufficiently homogeneous withrespect to each of at least three selected classification parameter (C₁,C₂, C₃, . . . C_(n)) values and sufficiently different from beads in anyother subset in at least one of said classification parameter values sothat the profile of classification parameter values within each subsetdetectable by flow cytometry is unique; and (2) are coupled to areactant that will specifically react with the analyte of interest inthe sample to be tested; and wherein the beads in a plurality of saidsubsets are coupled to the same reactant but at concentrations whichdiffer among said subsets; (b) mixing, to produce a reacted bead sample,the beadset with a fluorescently labeled competitive inhibitor of thereaction between the analyte of interest and the reactant on the beadsand with the fluid sample under conditions that will allow reactionsbetween the analyte of interest in the fluid sample and the reactants onthe beads in said set, wherein a reaction between an analyte of interestand a reactant on a bead causes a decrease in the value of a fluorescentsignal (F_(m)) emitted from said bead; (c) analyzing the reacted sampleby flow cytometry to determine the classification parameter valueprofile and an F_(m) value of each bead analyzed; (d) identifying thesubset to which each bead belongs and therefore the concentration ofreactant with which the bead was coupled as a function of the uniqueprofile of classification parameter values; (e) assigning a bead subsetvalue to each bead subset with correlates relatively with theconcentration of analyte with which the bead subset was coupled; (f)determining an inter-bead subset slope from a plot of mean F_(m) foreach bead subset versus bead subset value to produce an inter-beadsubset slope; and (g) determining the concentration of the analyte ofinterest in the sample by interpolation of the slope determined in step(f) into a standard assay curve wherein the inter-bead subset slopes ofbeads incubated with known concentrations of the analyte of interest areplotted against the log of the known concentration of the analyte ofinterest.
 52. A method of generating a multiplexed standard assay curvefor use in quantitating the concentration of an analyte of interest in afluid sample comprising the steps of: (a) obtaining a beadset comprisinga plurality of subsets of beads wherein the beads in said subset; (1)are sufficiently homogeneous with respect to each of at least threeselected classification parameter (C₁, C₂, C₃, . . C_(n)) values andsufficiently different from beads in any other subset in at least one ofsaid classification parameter values so that the profile ofclassification parameter values within each subset detectable by flowcytometry is unique; and (2) are coupled to a reactant that willspecifically react with a selected analyte of interest in a fluid to betested; and (3) wherein the beads in a plurality of said subsets arecoupled to the same reactant but at concentrations which differ amongsaid subsets; (b) mixing, to produce a reacted bead sample, the beadsetwith a fluorescently labeled competitive inhibitor of the analyte ofinterest and a known concentration of the analyte of interest underconditions that will allow reactions between the analyte of interest inthe fluid and the reactants on the beads in said set, wherein a reactionbetween a reactant and an analyte of interest on a bead causes adecrease in the value of a fluorescent signal (F_(m)) emitted from saidbead; (c) analyzing the reacted sample by flow cytometry to determinethe classification parameter value profile and an F_(m) value of eachbead analyzed; (d) identifying the subset to which each bead belongs andtherefore the concentration of reactant with which the bead was coupledas a function of the unique profile of classification parameter values;and (e) assigning a bead subset value to each bead subset withcorrelates relatively with the concentration of analyte with which thebead subset was coupled; and (f) determining an inter-bead subset slopefrom a plot of mean F_(m) for each bead subset versus bead subset value;and (g) repeating steps (a)-(f) at least one time but with a knownconcentration of analyte of interest that differs from saidconcentration of analyte of interest employed in any other step (b); and(h) plotting to produce a standard curve the inter-bead subset slopes ateach known concentration of analyte of interest against the log of eachknown concentration of analyte of interest.
 53. A method for flowcytometric analysis to detect a plurality of nucleic acid analytes ofinterest in a single sample comprising: (a) obtaining a beadsetcomprising a plurality of subsets of beads wherein the beads in eachsubset; (1) are sufficiently homogeneous with respect to each of atleast three selected classification parameter (C₁, C₂, C₃ . . . C_(n))values and sufficiently different from beads in any other subset in atleast one of said classification parameter values so that the profile ofclassification parameter values within each subset detectable by flowcytometry is unique, (2) are coupled to a nucleic acid that willspecifically hybridize with a selected nucleic acid analyte of interestin a fluid to be tested, (3) are reactive with a fluorescently labelednucleic acid probe which competes with said nucleic acid analyte forhybridization with said nucleic acid coupled to the bead; (b) mixing, toproduce a reacted bead sample, the beadset with the fluid to be testedunder conditions that will allow hybridization between nucleic acidanalytes of interest in the fluid and the nucleic acids coupled to thebeads in said beadset and thereby to allow the nucleic acid analytes insaid fluid to inhibit hybridization between the fluorescently labelednucleic acids with the nucleic acids coupled to said beads, resulting ina decrease in a fluorescent signal F_(m) emitted from a bead with whicha nucleic acid analyte of interest in the fluid has reacted; (c)analyzing the reacted sample by flow cytometry to determine theclassification parameter value profile and an F_(m) value of each beadanalyzed; (d) identifying the subset to which each bead belongs andtherefore the reactant on the bead as a function of the unique profileof classification parameter values; and (e) detecting the presence orabsence of a particular analyte of interest in said sample as a functionof the identification in step (d) and an increase in the F_(m) values ofthe beads in each of said subsets in the reacted fluid sample from theF_(m) values of the beads in each of said subsets not reacted with saidfluid.
 54. The method of claim 3 wherein said analytes are enzymes, saidreactants are fluorescent molecules which upon reaction with the enzymelose fluorescence, said change in F_(m) results from alteration of saidsubstrates attached to said beads.
 55. The method of claim 3 whereinsaid analytes are enzymes, said reactants are non-fluorescent moleculeswhich upon reaction with the enzyme become fluorescent, and said changein F_(m) results from alteration of said substrates attached to saidbeads.
 56. The method of claim 3 wherein said analytes are convertaseswhich produce active enzymes from inactive precursors, said reactantsare inactive precursors that are converted to active enzyme which inturn are reactants of fluorescently labeled substrates for said newlyactivated enzymes, and said change in F_(m), results from cleavage ofsaid substrates from said beads.
 57. The method of claim 5 wherein saidanalytes are enzymes, said reactants are molecules attached to a beadwhich, upon reaction with the enzyme, become ligates for a fluorescentlylabeled ligand, and wherein said change in F_(m) results from reactionof the new ligate with the fluorescently labeled ligand.
 58. The methodof claim 3 wherein said analyte is a cofactor which produces an activeenzyme from an inactive apo-enzymes, said reactant is a fluorescentlylabeled substrate for said activated enzyme, and said change in F_(m)results from cleavage of said substrate from said active enzyme.
 59. Amethod of processing a plurality of data signals generated by a flowcytometer in real-time, each said data signal being associated with aspecific cytometric target and encoding a forward light scatter value, aside light scatter value, a red fluorescence value, an orangefluorescence value, and a green fluorescence value, comprising: (a)receiving a data signal; (b) extracting from said data signal (1) aforward light scatter value, (2) a side light scatter value, (3) a redfluorescence value, (4) an orange fluorescence value, and (5) a greenfluorescence value; (c) classifying said cytometric target into one of aplurality of classes, referred to as an identified class, saidclassification being a function of said extracted (1) forward lightscatter value, (2) side light scatter value, (3) red fluorescence value,and (4) orange fluorescence value; (d) incrementing a class-count valueassociated with said identified class, said class-count value encodingthe number of cytometric targets classified as belonging to saididentified class; (e) accumulating a green-fluorescence-sum valueassociated with said identified class, said green fluorescence sum-valueencoding an arithmetic sum of said extracted green fluorescence valuefor all cytometric targets classified as belonging to said identifiedclass; (f) repeating the operations described in paragraphs (a) through(e) for subsequent data signals; (g) generating, for each of saidplurality of classes, one or more outcome-description signals encodingtextual information correlated with the class-count value and with thegreen-fluorescence-sum value for said class; and (h) displaying saidtextual information.
 60. The method of claim 59 wherein said specificcytometric target is an appropriately labeled bead.
 61. The method ofclaim 60 wherein each one of said plurality of classes is associatedwith one bead subset, said bead subset formed in accordance with claim2.
 62. The method of claim 59 wherein the operation of paragraph © isfurther comprised of performing a reasonableness test on said cytometrictarget's identified class, said reasonableness test being a function ofone or more of said identified class' (1) forward light scatter value,(2) side light scatter value, (3) red fluorescence value, and (4) orangefluorescence value.
 63. A method of processing a plurality of datasignals generated by a flow cytometer, each said data signal beingassociated with a specific flow cytometric target and encoding aplurality of classification parameter values and one or more measurementparameter values, comprising: (a) receiving a data signal; (b)extracting said plurality of classification parameter values and saidone or more measurement parameter values from said data signal; (c)classifying said cytometric target into one of a plurality of classes,referred to as an identified class, said classification being a functionof said plurality of extracted classification parameter values; (d)incrementing a class-count value associated with said identified class;(e) accumulating each of said one or more extracted measurement valuesinto one or more respective accumulation-values for said identifiedclass; (f) repeating the operations described in paragraphs (a) through(e) for subsequent data signals; (g) generating, for each of saidplurality of classes, one or more outcome-description signals encodinginformation correlated with the class-count and with the one or moreaccumulation-sum values for said class; and (h) displaying said textualinformation.
 64. The method of claim 63 wherein said processing inperformed in real-time.
 65. The method of claim 64 wherein each one ofsaid plurality of classes is associated with one bead subset, said beadsubset formed in accordance with claim
 2. 66. The method of claim 63wherein said specific cytometric target is an appropriately labeledbead.
 67. The method of claim 63 wherein said data signal encodes aplurality of classification parameter values selected from the groupconsisting of forward light scatter, side light scatter, redfluorescence, and orange fluorescence.
 68. The method of claim 63wherein said data signal encodes one or more measurement parametervalues selected from the group consisting of orange fluorescence andgreen fluorescence.
 69. The method of claim 63 wherein said one or moreoutcome-description signals encodes textual information.
 70. The methodof claim 63 wherein each of said one or more outcome-description signalsis determined by either an OVER-UNDER test or a SHIFT test.
 71. Anmachine readable assay database, stored in a storage device, for theprocessing of flow-cytometric measurement data comprising: (a) an assaydefinition table, said assay definition table encoding (1) one or moremeasurement subset token identifiers, (2) for each subset tokenidentifier, one or more baseline measurement parameter values, and (3)for each subset token identifier, an interpretation test-type token; (b)a discriminant function table, said discriminant function table encodinga classification decision tree based on one or more classificationmeasurement parameters, said one or more classification measurementparameters encoded in said flow-cytometric measurement data; (c) aninterpretation table, said interpretation table encoding textual assayoutcome-description information; and (d) a results table, said resultstable capable of encoding statistical accumulation of real-timeflow-cytometric measurement data.
 72. A method of processing a pluralityof data signals, in real-time, generated by a diagnostic device, each ofsaid plurality of data signals being associated with a specificdiagnostic target and encoding a plurality of classification parametervalues and one or more measurement parameter values, comprising: (a)receiving a data signal; (b) extracting said plurality of classificationparameter values and said one or more measurement parameter values fromsaid data signal; (c) classifying said diagnostic target into one of aplurality of classes, referred to as an identified class, saidclassification being a function of said plurality of extractedclassification parameter values; (d) incrementing a class-count valueassociated with said identified class; (e) accumulating each of said oneor more extracted measurement values into one or more respectiveaccumulation-values for said identified class; (f) repeating theoperations described in paragraphs (a) through (e) for subsequent datasignals; (g) generating, for each of said plurality of classes, one ormore outcome-description signals encoding information correlated withthe class-count and with the one or more accumulation-sum values forsaid class; and (h) displaying said outcome-description signals.
 73. Themethod of claim 72 wherein said diagnostic device is selected from thegroup consisting of a flow cytometer and a cell sorter.
 74. A programstorage device that is readable by a computer, said program storagedevice having encoded therein a program of instructions that includesinstructions for executing the method steps of a specified one of claims59, 63, 71, and
 72. 75. A method for flow cytometric analysis to detectgenetic mutations in a DNA comprising: (a) obtaining beads coupled to anoligonucleotide molecule designed to hybridize with a selected PCRproduct of interest; (b) mixing the beads with said PCR product underconditions that will allow hybridization between said PCR product andthe oligonucleotide coupled to the beads and thereby to allow the PCRproduct to inhibit hybridization between a fluorescently labeled nucleicacid probe that is completely complementary to said oligonucleotidecoupled to said beads; (c) adding said fluorescent probe to the mixture;(d) analyzing the reacted sample by flow cytometry to determine thefluorescence of each bead analyzed; and (e) detecting the geneticmutation or absence thereof as a result of the degree of fluorescence onthe beads.
 76. A method to detect a genetic mutations in a DNAcomprising: (a) obtaining beads coupled to an oligonucleotide molecule,said oligonucleotide molecule designed to hybridize with a selected PCRproduct of interest; (b) mixing said beads with said PCR product, underconditions that will allow hybridization between said PCR product andthe oligonucleotide coupled to the beads, to form a reacted mixture; (c)adding a fluorescent probe to said reacted mixture; (d) determining thefluorescence of the beads by flow cytometry; and (e) detecting thegenetic mutation, or absence thereof, as a result of the degree of thedetermined fluorescence on the beads.
 77. A method of detecting agenetic mutation in a DNA comprising the steps of: (a) selecting anoligonucleotide probe for said genetic mutation; (b) preparing afluorescent DNA probe complementary to the oligonucleotide probecoupling said selected probe to each one of a plurality of beads to forma bead aliquot; (c) selecting PCR primers to amplify a region of saidDNA corresponding to said selected probe; (d) amplifying said geneticmutation by PCR to form PCR products; (e) mixing said bead aliquot, saidPCR products and said fluorescent probe to form a mixture; (f)incubating said mixture to promote under competitive hybridizationconditions; (g) measuring the fluorescence said beads by flow cytometry;and (h) detecting said genetic mutation, or absence thereof, as afunction of the measured fluorescence of said beads.
 78. The method ofclaim 77 wherein said genetic mutation is selected from the groupconsisting of mutations in MEN2a, MEN2b, MEN1, ret proto-oncogene, LDLreceptor, NF1, NF type 2, BRCA1, BRCA2, BRCA3, APC, adenosine deaminase,XPAC, ERCC6 excision repair gene, fmr1, Duchenne's muscular dystrophygene, myotonic dystrophy protein kinase, androgen receptor,Huntington's, HPRT, apolipoprotein E, HEXA, steroid 2-hydroxylase,angiotensin, hNMLH1, 2 mismatch repair, APC, Rb, p53, bcr/abl, bcl-2gene, chromosomes 11 to 14 and chromosomes 15 to 17 gene transpositions,and genes encoding ion transporters.
 79. The method of claim 77 whereinsaid oligonucleotide probe has a length of between 5 and 500nucleotides.
 80. The method of claim 77 wherein said PCR primers aredesigned to amply a region of said DNA corresponding to saidoligonucleotide probe.
 81. The method of claim 77 wherein saidfluorescent probe is selected from the group consisting of DNA sequencescomplementary to wild-type or mutant sequences coupled to the beads. 82.A kit for detection of a genetic mutation in a DNA comprising: (a) afirst container comprising beads coupled to an oligonucleotide designedto hybridize with a selected PCR product of interest; (b) a secondcontainer comprising a PCR primer designed to amplify a section of DNAcomplementary to said oligonucleotide; and (c) a third containercomprising a fluorescent labeled DNA probe capable of selectivelyhybridizing said oligonucleotide.
 83. The kit of claim 82, wherein saidgenetic mutation is selected from the group consisting of mutations inMEN2a, MEN2b, MEN1, ret proto-oncogene, LDL receptor, NF1, NF type 2,BRCA1, BRCA2, BRCA3, APC, adenosine deaminase, XPAC, ERCC6 excisionrepair gene, fmr1, Duchenne's muscular dystrophy gene, myotonicdystrophy protein kinase, androgen receptor, Huntington's, HPRT,apolipoprotein E, HEXA, steroid 2-hydroxylase, angiotensin, hNMLH1, 2mismatch repair, APC, Rb, p53, bcr/abl, bcl-2 gene, chromosomes 11 to 14and chromosomes 15 to 17 gene transpositions, and genes encoding iontransporters.
 84. The kit of claim 82 wherein said fluorescent labeledDNA probe has a length of between 5 and 500 nucleotides.
 85. The methodclaim 3 where the analytes of interest are DNA segments, the reactant onthe bead are DNA segment capable of specifically hybridizing to saidanalytes, and the fluorescent label is a fluorescent DNA segment alsocapable of specifically hybridizing with said reactant to compete withthe hybridization of said reactant to said label.